Genotyping the human UDP-glucuronosyl transferase 1 (UGT1) gene

ABSTRACT

Genetic polymorphisms are identified in the human UGT1 gene that alter UGT1-dependent drug metabolism. Nucleic acids comprising the polymorphic sequences are used to screen patients for altered metabolism for UGT1 substrates, potential drug-drug interactions, and adverse/side effects, as well as diseases that result from environmental or occupational exposure to toxins. The nucleic acids are used to establish animal, cell and in vitro models for drug metabolism.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of earlier filed U.S. Provisional Application Serial No. 60/084,807, filed May 7, 1998.

INTRODUCTION

[0002] The metabolic processes commonly involved in the biotransformation of xenobiotics have been classified into functionalization reactions (phase I reactions), in which lipophilic compounds are modified via monooxygenation, dealkylation, reduction, aromatization, or hydrolysis. These modified molecules can then be substrates for the phase II reactions, often called conjugation reactions, as they conjugate a functional group with a polar, endogenous compound. Drug glucuronidation, a major phase II conjugation reaction in the mammalian detoxification system, is catalyzed by the UDP-glucuronosyltransferases (UGTs) (Batt A M, et al. (1994) Clin Chim Acta 226:171-190; Burchell et al. (1995) Life Sci. 57:1819-31).

[0003] The UGTs are a family of enzymes that catalyze the glucuronic acid conjugation of a wide range of endogenous and exogenous substrates including phenols, alcohols, amines and fatty acids. The reactions catalyzed by UGTs permit the conversion of a large range of toxic endogenous/xenobiotic compounds to more water-soluble forms for subsequent excretion (Parkinson A (1996) Toxicol Pathol 24:48-57).

[0004] The UGT isoenzymes are located primarily in hepatic endoplasmic reticulum and nuclear envelope (Parkinson A (1996) Toxicol Pathol 24:48-57), though they are also expressed in other tissues such as kidney and skin. UGTs are encoded by a large multigene superfamily that has evolved to produce catalysts with differing but overlapping substrate specificities. Three families, UGT1, UGT2, and UGT8, have been identified within the superfamily. UGTs are assigned to one of the subfamilies based on amino acid sequence identity, e.g., UGT1 family members have greater than 45% amino acid sequence identity (Mackenzie et al. 1997) Pharmacogenetics 7:255-69).

[0005] The UGT1 locus is located on chromosome 2q37, and contains at least 12 promoters/first exons, which are apparently able to splice with common exons 2 through 5, producing gene products having strikingly different N-terminal halves (amino acid sequence identities ranging from 24% to 49%), but identical C-terminal halves (FIG. 1). At least eight different isoenzymes are encoded by the UGT1 locus; at least one or more first exons encode pseudogenes. The different N-terminal halves encoded by the first exons confer different substrate binding specificities upon the UGT1 isoenzymes, while exons 2-5, which are present in all UGT1 isoenzyme mRNAs, encode the UDP-glucuronic acid binding domain, membrane anchorage site, and ER retention signal that are common to all UGT proteins (Ritter et al. (1992) J Biol Chem 267:3257-3261). UGT1 locus isoenzymes are best known for their role in glucoronidation and metabolism of many substrates, including bilirubin (1A1, 1D1), planar and non-planar phenols, naphthols (1F1) (Ouzzine M, et al. (1994) Arch Biochem Biophys 310:196-204), anthraquinones, flavones, aliphatic alcohols, aromatic carboxylic acids, and steroids (Ebner T, et al. (1993) Drug Metab Dispos 21:50-55).

[0006] In addition to UGT1 exon usage, metabolism of endogenous and exogenous substrates can also be affected by competitive binding phenomena. For example, in some cases exogenous substrates for the UGT1 enzymes have a higher binding affinity or avidity for the enzyme than the endogenous UGT1 substrates. For example, UGT1*1, the major bilirubin-metabolizing form of UGT1, more readily binds both octyl-gallate and emodin than it binds bilirubin, thus indicating the potential of these xenobiotics to cause jaundice by inhibition of bilirubin binding to UGT1*1 (where 1*1 indicates that the first exon is used in the spliced gene product). UGT1*1 is also responsible for glucuronidation of the oral contraceptive ethinylestradiol (Ebner et al. (1993) Mol. Pharmacol. 43:649-54), and can also glucuronidate phenols, anthroquinones, flavones, and certain endogenous steroids.

[0007] As noted above, the first exon present in UGT1 can affect substrate binding specificity of the UGT1 gene product (for a review, see Burchell (1995) Life Sci. 57:1819-31). For example, UGT *2 accepts a wide range of compounds as substrates including non-planar phenols, anthraquinones, flavones, aliphatic alcohols, aromatic carboxylic acids, steroids (4-hydroxyestrone, estrone) and many drugs of varied structure (Ebner et al. (1993) Drug. Metab. Disp. 21:50-5; Burchell (1995) Life Sci. 57:1819-31). In contrast, UGT1*6 exhibits only limited substrate specificity for planar phenolic compounds relative to other human UGTs.

[0008] Polymorphisms can markedly affect binding of the endogenous substrate, which can be manifested as clinical syndromes. At least two conditions, Crigler-Najjar syndrome and Gilbert syndrome, are associated with UGT1 polymorphisms. Both of these syndromes are hereditary and are associated with predominantly unconjugated hyperbilirubinemia. Crigler-Najjar syndrome is associated with intense, persistent jaundice which begins at birth. Some affected infants die in the first weeks or months of life with kernicterus; others survive with little or no neurologic defect. Crigler-Najjar syndrome is caused by a defect in the ability of UGT1 to catalyze UDP-glucuronidation of bilirubin, resulting in accumulation of bilirubin in the blood (Erps et al. (1994) J. Clin. Invest. 93:564-70). Gilbert syndrome is a benign mild form of unconjugated hyperbilirubinemia that is characterized by normal liver function tests, normal liver histology, delayed clearance of bilirubin from the blood, and mild jaundice that tends to fluctuate in severity. As with Crigler-Najar syndrome, Gilbert syndrome is associated with a defect in UGT1. Specific UGT polymorphisms that are known to be associated with disease are indicated in FIG. 1.

[0009] Alteration of the expression or function of UGTs may also affect drug metabolism. For example, there may be common polymorphisms in the human UGT1 gene that alter expression or function of the protein product and cause drug exposure-related phenotypes. Thus, there is a need in the field to identify UGT1 polymorphisms in order to provide a better understanding of drug metabolism and the diagnosis of drug exposure-related phenotypes.

Relevant Literature

[0010] Genbank accession number M84122 provides UGT1 exon 2, M84123 provides exons 3 and 4, M84124 provides 5, M84125 provides exon 1A, M84127 provides exon 1C, M84128 provides exon 1D, M84129 provides exon 1E, M84130 provides exon 1F, U39570 provides exon 1 G, U42604 provides exon 1H, U39550 provides exon 1J.

[0011] The UGT gene superfamily and recommended nomenclature for describing UGT genes and alleles are reviewed in Mackenzie et al. (1997) Pharmacogenet. 7:255-69.

[0012] The two UGT1A6 genetic polymorphisms are described in Ciotti et al. (1997) Am. J. Hum. Genet. 61(Supp):A249. The identification of Asp446 as a critical residue in UGT1 is described in Iwano et al. (1997) Biochem. J. 325:587-91.

[0013] A review of the substrate specificity of human UDP-glucuronosyltransferases is provided by Burchell et al. (1995) Life Sci. 57:1819-31. For a review of drug glucoronidation in humans, see Miners et al. (1991) Pharmacol. Ther. 51:347-69.

[0014] At least twelve UGT1 μl polymorphisms have been identified and linked to disease. These UGT1A1 alleles, each of described in OMIM Entry 191740 (at http://www.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?191740) and in OMIM Entry 143500 (at http://www.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?143500), include:

[0015] 1) UGT1*FB (UGT1A1, 13-BP DEL, EX2; 191740.0001), which contains a 13 bp deletion in exon 2 and is associated with Crigler-Najjar syndrome type I (CN-I);

[0016] 2) UGT1A1, EXON4, C-T, SER-PHE (191740.0002), which contains a C-to-T transition in exon 4 (resulting in an amino acid change from serine to phenylalanine) is associated with CN-I and deficiency of both bilirubin-UGT and phenol-UGT activities in the liver;

[0017] 3) UGT1A1, GLN331TER (191740.0003), which contains a C-to-T transition 6 bp upstream from the 3-prime end of exon 2 of the common region (replacement of a glutamine codon with a stop codon), is associated with CN-I;

[0018] 4) UGT1 Al, ARG341TER (191740.0004), which contains a nonsense mutation (CGA-to-TGA) in exon 3 and is associated with CN-I and a total absence of all phenol/bilirubin UGT proteins and their activities in liver homogenate by enzymologic and immunochemical analysis;

[0019] 5) UGT A1, GLN331 ART (191740.0005), which contains an A-to-G transition bp upstream of the exon 2/intron 2 boundary (resulting in a glutamine-to-arginine substitution), is associated with Crigler-Najjar Syndrome, type II (CN-II);

[0020] 6) UGT A1, PHE170DEL (191740.0006), which contains a deletion of the phenylalanine codon at position 170 in exon 1, and is associated with CN-I;

[0021] 7) UGT A1, SER376PHE (191740.0007), which contains a C-to-T transition in codon 376 (resulting in a change of serine to phenylalanine) and is associated with CN-1;

[0022] 8) UGTLA1, GLY309GLU (191740.0008), which contains a G-to-A transition in codon 309 (resulting in a glycine to glutamic acid change) and is associated with CN-I;

[0023] 9) UGT A1, NT840, C-A, CYS-TER (191740.0009), which contains a C-to-A transversion at base position 840 in exon 1 (resulting in replacing a cysteine with a stop codon), is associated with CN-I;

[0024] 10) UGT1A1, PRO229GLN (191740.00010), which contains C-to-A transversion at nucleotide 686 (changing proline-229 to glutamine), is associated with Gilbert syndrome;

[0025] 11) UGT1A1, 2-BP INS, TA INS, TATM ELEMENT (191740.00011) contains 2 extra bases (TA) in the TATAA element of the 5-prime promoter region of the gene (where normally an A(TA)6TAA element is present between nucleotides −23 and −3) and is associated with Gilbert syndrome; and

[0026] 12) UGT1A1, 1-BP INS, 470T INS (191740.00012), which contains 470insT mutation in exon 1 and is associated with CN-I.

SUMMARY OF THE INVENTION

[0027] Genetic sequence polymorphisms are identified in the UGT1 gene. Nucleic acids comprising the polymorphic sequences are used in screening assays, and for genotyping individuals. The genotyping information is used to predict an individuals' rate of metabolism for UGT1 substrates, potential drug-drug interactions, and adverse/side effects.

[0028] Accordingly, in one aspect the invention features an isolated nucleic acid molecule comprising a UGT1 sequence polymorphism of SEQ ID NOS:87-124, as part of other than a naturally occurring chromosome. In related aspects, the invention features nucleic acid probes for detection of UGT1 locus polymorphisms, where the probe comprises a polymorphic sequence of SEQ ID NOS:87-124.

[0029] In another aspect the invention features an array of oligonucleotides comprising two or more probes for detection of UGT1 locus polymorphisms, where the probes comprise at least one form of a polymorphic sequences of SEQ ID NOS:87-124.

[0030] In still another aspect, the invention features a method for detecting in an individual a polymorphism in UGT1 metabolism of a substrate, where the method comprises analyzing the genome of the individual for the presence of at least one UGT1 polymorphism of SEQ ID NOS:87-124; wherein the presence of the predisposing polymorphism is indicative of an alteration in UGT1 expression or activity.

[0031] In one embodiment, the analyzing step of the method is accomplished by detection of specific binding between the individual's genomic DNA with an array of oligonucleotides comprising two or more probes for detection of UGT1 locus polymorphisms, where the probes comprise at least one form of a polymorphic sequence of SEQ ID NOS:87-124.

[0032] In other embodiments of the method, the alteration is UGT1 expression or activity is tissue specific, or is in response to a UGT1 modifier. The UGT1 modifier may either induce or inhibit UGT1 expression.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 is a schematic showing the UGT1 locus. Each of the first exons is denoted by both its alphabetic and numerical nomenclatures (e.g., 1A and 1.1).

[0034]FIG. 2 is a schematic showing exons 1A-1J of the UGT1 locus and the polymorphisms described in the present application.

[0035]FIG. 3 is a schematic showing the exons 1A-1F, and 2-5 of the UGT1 locus and the polymorphisms that have been publicly disclosed.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0036] UGT1 Reference Sequences. SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, and 15 are the UGT1 reference polynucleotide sequences for UGT1 exons 1A, 1C, 1D, 1E, 1F, 1G, 1H, and 1J. The polypeptide sequences are encoded by these reference exon sequences are SEQ ID NOS:2, 4, 6, 8, 12, 14, and 16. SEQ ID NOS: 17 and 18 are the reference polynucleotide and amino acid sequences for UGT1 exons 2-5.

[0037] PCR Primers. The primary and secondary PCR primers for amplification of polymorphic sequences are presented as SEQ ID NOS:19-50.

[0038] Sequencing Primers. The primers used in sequencing isolated polymorphic sequences are presented as SEQ ID NOS:51-86.

[0039] Polymorphisms. Polymorphic sequences of the invention are presented as SEQ ID NOS:88-124.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0040] Pharmacogenetics is the linkage between an individual's genotype and that individual's ability to metabolize or react to a therapeutic agent. Differences in metabolism or target sensitivity can lead to severe toxicity or therapeutic failure by altering the relation between bioactive dose and blood concentration of the drug. Relationships between polymorphisms in metabolic enzymes or drug targets and both response and toxicity can be used to optimize therapeutic dose administration.

[0041] Genetic polymorphisms are identified in the UGT1 gene. Nucleic acids comprising the polymorphic sequences are used to screen patients for altered metabolism for UGT1 substrates, potential drug-drug interactions, and adverse/side effects, as well as diseases that result from environmental or occupational exposure to toxins. The nucleic acids are used to establish animal, cell culture and in vitro cell-free models for drug metabolism.

Definitions

[0042] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0043] As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a construct” includes a plurality of such constructs and reference to “the UGT1 nucleic acid” includes reference to one or more nucleic acids and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

[0044] UGT1 polymorphic sequences. The sequence of the UGT1 gene is known in the art, and accessible in public databases, as cited above. This sequence is useful as a reference for the genomic location of the human gene, and for specific coding region sequences. As used herein, the term “UGT1 gene” is intended to refer to both the wild-type and variant sequences, unless specifically denoted otherwise. Nucleic acids of particular interest comprise the provided variant nucleotide sequence(s). For screening purposes, hybridization probes may be used where both polymorphic forms are present, either in separate reactions, or labeled such that they can be distinguished from each other. Assays may utilize nucleic acids that hybridize to one or more of the described polymorphisms.

[0045] The genomic UGT1 sequence is of particular interest. A polymorphic UGT1 gene sequence, i.e. including one or more of the provided polymorphisms, is useful for expression studies to determine the effect of the polymorphisms on enzymatic activity. The polymorphisms are also used as single nucleotide polymorphisms to detect genetic association with phenotypic variation in UGT1 activity and expression.

[0046] The UGT1 exon structure is illustrated in FIG. 1. The UGT1 locus contains at least 12 promoters/first exons, which are apparently able to splice with common exons 2 through 5, producing gene products having different N-terminal halves but identical C-terminal halves. The first exon utilized at least in part determines the substrate specificity of the resulting UGT1 gene product. Each of the first exons in FIG. 1 is denoted by both its alphabetic and numerical nomenclatures (e.g., 1A and 1.1). Polymorphisms in the UGT1 first exon can be associated with alteration of substrate binding specificity and/or disease. FIG. 2 shows UGT1 exons 1A-1J and the polymorphisms described in the present application. FIG. 3 shows UGT1 exons 1 A-1 F and 2-5 and the polymorphisms in these exons that have been publicly disclosed. Polymorphisms denoted by an asterisk (*) have been assigned the indicated “allele name” (e.g., *12). The specific associated disease is indicated below in parentheses for several of these disease-associated polymorphisms. Except for the “mutation” that is associated with Gilbert's (*28, which is not universally agreed upon in the literature), all mutations in exons 1 D, 1 A, and 2-5 were isolated from individuals with disease.

[0047] Fragments of the DNA sequence are obtained by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, DNA fragments will be of at least 15 nt, usually at least 20 nt, often at least 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than 100 nt are useful for production of the encoded polypeptide, promoter motifs, etc. For use in amplification reactions, such as PCR, a pair of primers will be used. The exact composition of primer sequences is not critical to the invention, but for most applications the primers will hybridize to the subject sequence under stringent conditions, as known in the art.

[0048] The UGT1 nucleic acid sequences are isolated and obtained in substantial purity, generally as other than an intact mammalian chromosome. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include a UGT1 sequence or fragment thereof, generally being at least about 50%, usually at least about 90% pure and are typically “recombinant”, i.e. flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.

[0049] UGT1 polypeptides. The UGT1 genetic sequence, including polymorphisms, may be employed for synthesis of a complete UGT1 protein, or polypeptide fragments thereof, particularly fragments corresponding to functional domains; binding sites; etc.; and including fusions of the subject polypeptides to other proteins or parts thereof. For expression, an expression cassette may be employed, providing for a transcriptional and translational initiation region, which may be inducible or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. Various transcriptional initiation regions may be employed that are functional in the expression host. The polypeptides may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. Small peptides can also be synthesized in the laboratory.

[0050] Substrate. A substrate is a chemical entity that is modified by UGT1, usually under normal physiological conditions. Although the duration of drug action tends to be shortened by metabolic transformation, drug metabolism is not “detoxification”. Frequently the metabolic product has greater biologic activity than the drug itself. In some cases the desirable pharmacologic actions are entirely attributable to metabolites, the administered drugs themselves being inert. Likewise, the toxic side effects of some drugs may be due in whole or in part to metabolic products.

[0051] Substrates can be either endogenous substrates (e.g., substrates normally found within the natural environment of UGT1, such as the bilirubin or other endobiotic compound) or exogenous (e.g., substrates that are not normally found within the natural environment of UGT1, such as ethinyl estradiol or other xenobiotic compound). Exemplary UGT1 substrates (i.e., substrates of wild-type UGT1 and/or UGT1 polypeptides encoded by UGT1 polymorphisms) include, but are not necessarily limited to endobiotics such as bilirubin, bilirubin monoglucoronide, bile acids, steroids, thyroxine, biogenic amines, fat-soluble vitamins, UDPGA, 17β estradiol, estriol, 2-hydroxy-estriol, T4,rT3, and the like; and xenobiotics such as hydroxylated polycyclic aromatic hydrocarbons, heterocyclics, carcinogens, plant metabolites, octyl gallate, ethinylestradiol, anthraflavic acid, quercetin, 1-naphthol, naphthylamines, 4-aminobiphenyl, benzidine, imipamine, BP-3,6-quinol, 5-hydroxy-BP, acetaminophen, vanillin, naproxen, 4-methylumbelliferone, monohalogenated phenols, propofol, 4t-pentylphenol, 4-hydroxybiphenyl, carvacrol, emodin, galangin, bulky phenols, carboxylic acids, 5-hydroxy 2AAF, 8-hydroxy 2AAF, and the like. Table 1 provides a summary of the major endobiotic and xenobiotic substrates, as well as exemplary non-substrates, of four UGT1 isoenzymes (UGT1*1 (same as UGT1A), UGT1*4 (same as UGT1D), UGT1*6 (same as UGT1F), and UGT1*02 (same as UGT1 G) (see Burchell et al. 91995) Life Sci. 57:1819-31). TABLE 1 Substrate Specificity of Human Liver UGT1 Isoenzymes Isoenzyme Endobiotic Xenobiotic Non-substrate UGT1*1 Bilirubin (Km 24 pm) Octyl gallate (Km 162 μm) Gallic acid Bilirubin monoglucuronide Ethinylestradiol T3 UDPGA (Km 0.41 mM) Anthraflavic acid Menthol 17β estradiol Quercetin Retinoic acid Estriol 1-naphthol Clofibrate 2-hydrozy-estriol Morphine T4, rT3 Propofol Testosterone UGT1*4 Bilirubin? Naphthylamines Bilirubin? 4-aminobiphenyl Carbamazepine Benzide Imipamine UGT1*6 1-Naphthol 4-Hydroxybiphenyl BP-3,6-quinol Propofol 5-hydroxy-BP Galangin Acetaminophen (Km 2 mM) Emodin Vanillin Morphine Naproxen Estriol 4-methylumbelliferone Estradiol Monohalogenated phenols AZT Menthol UGT1*7 UDPGA (Km 0.41 mM) Propofol (Km 172 μm) Morphine T4, rT3 4t-pentylphenol Estriol 4-hydroxybiphenyl Estradiol Carvacrol AZT Emodin Menthol Galangin Chloramphenicol Octyl gallate (Km 158 μM) Androsterone Other bulky phenols T3 Acetaminophen (Km 50 mM) Carboxylic acids (some) 5-hydroxy 2AAF 8-hydroxy 2AAF

[0052] Modifier. A modifier is a chemical agent that modulates the action of UGT1, either through altering its enzymatic activity (enzymatic modifier) or through modulation 10 of expression (expression modifier, e.g., by affecting transcription or translation). In some cases the modifier may also be a substrate. For example, the UGT1 gene contains an electrophile responsive element (U.S. Pat. No. 5,589,504); thus, compounds such as metabolites of planar aromatic compounds and phenolic antioxidants, as well as reactive oxygen species including peroxides would be expression modifiers via their effect on the electrophile responsive element. Endogenous and exogenous inducers that are capable of inducing particular UGT activities include phenobarbital, dioxin, peroxisome proliferators, rifamicin, oral contraceptive drug, carbamazepine, cigarette smoke, cabbage, brussel sprouts, polycyclic/aromatic hydrocarbons, and derivatives of indole 3-carbonil (see Burchell et al. (1995), supra, Parkinson In: “Biotransformation of Xenobiotics.” Chapter 6, Casarett & Doull's Toxicology, 5^(th) Ed., C. Klaassen, ed.)).

[0053] Pharmacokinetic parameters Pharmacokinetic parameters provide fundamental data for designing safe and effective dosage regimens. A drug's volume of distribution, clearance, and the derived parameter, half-life, are particularly important, as they determine the degree of fluctuation between a maximum and minimum plasma concentration during a dosage interval, the magnitude of steady state concentration and the time to reach steady state plasma concentration upon chronic dosing. Parameters derived from in vivo drug administration are useful in determining the clinical effect of a particular UGT1 genotype.

[0054] Expression assay. An assay to determine the effect of a sequence polymorphism on UGT1 expression. Expression assays may be performed in cell-free extracts, or by transforming cells with a suitable vector. Alterations in expression may occur in the basal level that is expressed in one or more cell types, or in the effect that an expression modifier has on the ability of the gene to be inhibited or induced. Expression levels of a variant alleles are compared by various methods known in the art. Methods for determining promoter or enhancer strength include quantitation of the expressed natural protein; insertion of the variant control element into a vector with a reporter gene such as -galactosidase, luciferase, chloramphenicol acetyltransferase, etc. that provides for convenient quantitation; and the like.

[0055] Gel shift or electrophoretic mobility shift assay provides a simple and rapid method for detecting DNA-binding proteins (Ausubel, F. M. et al. (1989) In: Current Protocols in Molecular Biology, Vol. 2, John Wiley and Sons, New York). This method has been used widely in the study of sequence-specific DNA-binding proteins, such as transcription factors. The assay is based on the observation that complexes of protein and DNA migrate through a nondenaturing polyacrylamide gel more slowly than free DNA fragments or double-stranded oligonucleotides. The gel shift assay is performed by incubating a purified protein, or a complex mixture of proteins (such as nuclear or cell extract preparations), with an end-labeled DNA fragment containing the putative protein binding site. The reaction products are then analyzed on a nondenaturing polyacrylamide gel. The specificity of the DNA-binding protein for the putative binding site is established by competition experiments using DNA fragments or oligonucleotides containing a binding site for the protein of interest, or other unrelated DNA sequences.

[0056] Expression assays can be used to detect differences in expression of polymorphisms with respect to tissue specificity, expression level, or expression in response to exposure to various substrates, and/or timing of expression during development. For example, since UGT1A and UGT1E are expressed in liver, UGT1A and UGT1E polymorphisms could be evaluated for expression in tissues other than liver, or expression in liver tissue relative to a reference UGT1 A or UGT1E polypeptide. Similarly, expression of polymorphisms in UGT1 F, which is normally expressed in liver, kidney and skin, could be assayed in each of these tissues and the relative levels of expression compared to a reference UGT1 F polypeptide.

[0057] Substrate screening assay. Substrate screening assays are used to determine the metabolic activity of a UGT1 protein or peptide fragment on a substrate. Many suitable assays are known in the art, including the use of primary or cultured cells, genetically modified cells (e.g., where DNA encoding the UGT1 polymorphism to be studied is introduced into the cell within an artificial construct), cell-free systems, e.g. microsomal preparations or recombinantly produced enzymes in a suitable buffer, or in animals, including human clinical trials (see, e.g., Burchell et al. (1995) Life Sci. 57:1819-1831, specifically incorporated herein by reference. Where genetically modified cells are used, since most cell lines do not express UGT1 activity (liver cells lines being the exception), introduction of artificial construct for expression of the UGT1 polymorphism into many human and non-human cell lines does not require additional modification of the host to inactivate endogenous UGT1 expression/activity. Clinical trials may monitor serum, urine, etc. levels of the substrate or its metabolite(s).

[0058] Typically a candidate substrate is input into the assay system, and the conversion to a metabolite is measured over time. The choice of detection system is determined by the substrate and the specific assay parameters. Assays are conventionally run, and will include negative and positive controls, varying concentrations of substrate and enzyme, etc.

[0059] Genotyping: UGT1 genotyping is performed by DNA or RNA sequence and/or hybridization analysis of any convenient sample from a patient, e.g. biopsy material, blood sample (serum, plasma, etc.), buccal cell sample, etc. A nucleic acid sample from an individual is analyzed for the presence of polymorphisms in UGT1, particularly those that affect the activity or expression of UGT1. Specific sequences of interest include any polymorphism that leads to changes in basal expression in one or more tissues, to changes in the modulation of UGT1 expression by modifiers, or alterations in UGT1 substrate specificity and/or activity.

[0060] Linkage Analysis: Diagnostic screening may be performed for polymorphisms that are genetically linked to a phenotypic variant in UGT1 activity or expression, particularly through the use of microsatellite markers or single nucleotide polymorphisms (SNP). The microsatellite or SNP polymorphism itself may not phenotypically expressed, but is linked to sequences that result in altered activity or expression. Two polymorphic variants may be in linkage disequilibrium, i.e where alleles show non-random associations between genes even though individual loci are in Hardy-Weinberg equilibrium.

[0061] Linkage analysis may be performed alone, or in combination with direct detection of phenotypically evident polymorphisms. The use of microsatellite markers for genotyping is well documented. For examples, see Mansfield et al. (1994) Genomics 24:225-233; and Ziegle et al. (1992) Genomics 14:1026-1031. The use of SNPs for genotyping is illustrated in Underhill et al. (1996) Proc Natl Acad Sci U S A 93:196-200.

[0062] Transgenic animals. The subject nucleic acids can be used to generate genetically modified non-human animals or site specific gene modifications in cell lines. The term “transgenic” is intended to encompass genetically modified animals having a deletion or other knock-out of UGT1 gene activity, having an exogenous UGT1 gene that is stably transmitted in the host cells, or having an exogenous UGT1 promoter operably linked to a reporter gene. Transgenic animals may be made through homologous recombination, where the UGT1 locus is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like. Of interest are transgenic mammals, e.g. cows, pigs, goats, horses, etc., and particularly rodents, e.g. rats, mice, etc.

[0063] Genetically Modified Cells. Primary or cloned cells and cell lines are modified by the introduction of vectors comprising UGT1 gene polymorphisms. The gene may comprise one or more variant sequences, preferably a haplotype of commonly occurring combinations. In one embodiment of the invention, a panel of two or more genetically modified cell lines, each cell line comprising a UGT2B4 polymorphism, are provided for substrate and/or expression assays. The panel may further comprise cells genetically modified with other genetic sequences, including polymorphisms, particularly other sequences of interest for pharmacogenetic screening, e.g. UGT1, other UGT2 sequences, cytochrome oxidase polymorphisms, etc.

[0064] Vectors useful for introduction of the gene include plasmids and viral vectors, e.g. retroviral-based vectors, adenovirus vectors, etc. that are maintained transiently or stably in mammalian cells. A wide variety of vectors can be employed for transfection and/or integration of the gene into the genome of the cells. Alternatively, micro-injection may be employed, fusion, or the like for introduction of genes into a suitable host cell.

Genotyping Methods

[0065] The effect of a polymorphism in the UGT1 gene sequence on the response to a particular substrate or modifier of UGT1 is determined by in vitro or in vivo assays. Such assays may include monitoring the metabolism of a substrate during clinical trials to determine the UGT1 enzymatic activity, specificity or expression level. Generally, in vitro assays are useful in determining the direct effect of a particular polymorphism, while clinical studies will also detect an enzyme phenotype that is genetically linked to a polymorphism.

[0066] The response of an individual to the substrate or modifier can then be predicted by determining the UGT1 genotype, with respect to the polymorphism. Where there is a differential distribution of a polymorphism by racial background, guidelines for drug administration can be generally tailored to a particular ethnic group.

[0067] The basal expression level in different tissue may be determined by analysis of tissue samples from individuals typed for the presence or absence of a specific polymorphism. Any convenient method may be use, e.g. ELISA, RIA, etc. for protein quantitation, northern blot or other hybridization analysis, quantitative RT-PCR, etc. for mRNA quantitation. The tissue specific expression is correlated with the genotype.

[0068] The alteration of UGT1 expression in response to a modifier is determined by administering or combining the candidate modifier with an expression system, e.g. animal, cell, in vitro transcription assay, etc. The effect of the modifier on UGT1 transcription and/or steady state mRNA levels is determined. As with the basal expression levels, tissue specific interactions are of interest. Correlations are made between the ability of an expression modifier to affect UGT1 activity, and the presence of the provided polymorphisms. A panel of different modifiers, cell types, etc. may be screened in order to determine the effect under a number of different conditions.

[0069] A UGT1 polymorphism that results in altered enzyme activity or specificity is determined by a variety of assays known in the art. The enzyme may be tested for metabolism of a substrate in vitro, for example in defined buffer, or in cell or subcellular lysates, where the ability of a substrate to be metabolized by UGT1 under physiologic conditions is determined. Where there are not significant issues of toxicity from the substrate or metabolite(s), in vivo human trials may be utilized, as previously described.

[0070] The genotype of an individual is determined with respect to the provided UGT1 gene polymorphisms. The genotype is useful for determining the presence of a phenotypically evident polymorphism, and for determining the linkage of a polymorphism to phenotypic change.

[0071] A number of methods are available for analyzing nucleic acids for the presence of a specific sequence. Where large amounts of DNA are available, genomic DNA is used directly. Alternatively, the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis. The nucleic acid may be amplified by conventional techniques, such as the polymerase chain reaction (PCR), to provide sufficient amounts for analysis. The use of the polymerase chain reaction is described in Saiki et al. (1985) Science 230:1350-1354, and a review of current techniques may be found in Sambrook et al. Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.14.2-14.33. Amplification may be used to determine whether a polymorphism is present, by using a primer that is specific for the polymorphism. Alternatively, various methods are known in the art that utilize oligonucleotide ligation as a means of detecting polymorphisms, for examples see Riley et al. (1990) Nucleic Acids Res 18:2887-2890; and Delahunty et al. (1996) Am J Hum Genet 58:1239-1246.

[0072] A detectable label may be included in an amplification reaction. Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels, e.g. 32P, 35S, 3H; etc. The label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.

[0073] The sample nucleic acid, e.g. amplified or cloned fragment, is analyzed by one of a number of methods known in the art. The nucleic acid may be sequenced by dideoxy or other methods. Hybridization with the variant sequence may also be used to determine its presence, by Southern blots, dot blots, etc. The hybridization pattern of a control and variant sequence to an array of oligonucleotide probes immobilized on a solid support, as described in U.S. Pat. No. 5,445,934, or in WO95/35505, may also be used as a means of detecting the presence of variant sequences. Single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), mismatch cleavage detection, and heteroduplex analysis in gel matrices are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility. Alternatively, where a polymorphism creates or destroys a recognition site for a restriction endonuclease (restriction fragment length polymorphism, RFLP), the sample is digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel or capillary electrophoresis, particularly acrylamide or agarose gels.

[0074] In one embodiment of the invention, an array of oligonucleotides are provided, where discrete positions on the array are complementary to one or more of the provided polymorphic sequences, e.g. oligonucleotides of at least 12 nt, frequently 20 nt, or larger, and including the sequence flanking the polymorphic position. Such an array may comprise a series of oligonucleotides, each of which can specifically hybridize to a different polymorphism. For examples of arrays, see Hacia et al. (1996) Nat Genet 14:441-447 and DeRisi et al. (1996) Nat Genet 14:457-460. Arrays of interest may further comprise sequences, including polymorphisms, of other genetic sequences, particularly other sequences of interest for pharmacogenetic screening, e.g. UGT1, other UGT2 sequences, cytochrome oxidase polymorphisms, etc.

[0075] The genotype information is used to predict the response of the individual to a particular UGT1 substrate or modifier. Where an expression modifier inhibits UGT1 expression, then drugs that are a UGT1 substrate will be metabolized more slowly if the modifier is co-administered. Where an expression modifier induces UGT1 expression, a co-administered substrate will typically be metabolized more rapidly. Similarly, changes in UGT1 activity will affect the metabolism of an administered drug. The pharmacokinetic effect of the interaction will depend on the metabolite that is produced, e.g. a prodrug is metabolized to an active form, a drug is metabolized to an inactive form, an environmental compound is metabolized to a toxin, etc. Consideration is given to the route of administration, drug-drug interactions, drug dosage, etc.

EXAMPLES

[0076] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

Example Identification of UGT1 Polymorphisms

[0077] Materials and Methods

[0078] DNA Samples. Blood specimens were collected from approximately 48 individuals after obtaining informed consent. All samples were stripped of personal identifiers to maintain confidentiality. Genomic DNA was isolated from these samples using standard techniques. Genomic DNA was stored either as a concentrated solution, or in a dried form in microtiter plates.

[0079] PCR amplifications. The primers used to amplify all exons are shown in Table 2, and were designed with NBI's Oligo version 5.0 program. TABLE 2 PCR Primers. (Ex = Exon) EX FORWARD PRIMER REVERSE PRIMER PRIMARY PCR AMPLIFICATION 1A TGGTGTATCGATTGGTTTT (SEQ ID NO:19) CATATATCTGGGGCTAGTTAATC (SEQ ID NO:20) 1C ACAAGGTAATTAAGATGAAGAAAGCA (SEQ ID NO:21) ACCTGAGATAGTGGCTTCCT (SEQ ID NO:22) 1D TTTGTCTTCCPATTACATGC (SEQ ID NO:23) AGTAGATATGGAAGCACTTGTAAG (SEQ ID NO:24) 1E TCTCAGTGACAACGTAATTAAGAC (SEQ ID NO:25) CATTGATTGGATAAAGGCA (SEQ ID NO:26) 1F AATTTGGGTTCTTACATATCAA (SEQ ID NO:27) GAGTGAGGGAGCACAGAG (SEQ ID NO:28) 1G ATAAGTACACGCCTTCTTTTG (SEQ ID NO:29) GCTGCTTTATACAATTTGCTAC (SEQ ID NO:30) 1H CGCCTACGTATCATAGCAGTTA (SEQ ID NO:31) GGAAAGAAATTTGAAATGCAAC (SEQ ID NO:32) 1J TCTTTCCGCCTACTGTATCA (SEQ ID NO:33) TTCAAGAAGGGCAGTTTTAT (SEQ ID NO:34) SECONDARY PCR AMPLIFICATION 1A CTCTGGCAGGAGCAAAG (SEQ ID NO:35) ATACACACCTGGGATAGTGG (SEQ ID NO:36) 1C GGTAATTAAGATGAAGAAAGCA (SEQ ID NO:37) CTGAGATAGTGGCTTCCTG (SEQ ID NO:38) 1D GTGGCTCAATGACAAGG (SEQ ID NO:39) ATATGGAAGCACTTGTAAGTAAA (SEQ ID NO:40) 1E TTAAGACGAAGGPAACAATTCT (SEQ ID NO:41) ACCTGAGATAGTGGCTTCC (SEQ ID NO:42) 1F ATCAAAGGGTAAAATTCAGA (SEQ ID NO:43) GGCAGTCCAAAAGAAATA (SEQ ID NO:44) 1G TTTTGAGGGCAGGTTCTA (SEQ ID NO:45) AATGGGACAAATGTAAATGATA (SEQ ID NO:46) 1H TTCTCTCATGGCTCGCA (SEQ ID NO:47) ATGTCAAATCACAATTCAGTAAGG (SEQ ID NO:48) 1J CCGCCTACTGTATCATAGCA (SEQ ID NO:49) CAACGAAATGTCAAATCACAG (SEQ ID NO:50)

[0080] Publicly available genomic sequences were used as references. Twenty-five nanograms of genomic DNA were amplified in the primary amplifications using the Perkin Elmer GeneAmp PCR kit according to the manufacturer's instructions in 25 μl reactions with AmpliTaq Gold DNA polymerase. Reactions contained 25 mM MgCl₂ and 0.2 μM of each primer. Thermal cycling was performed using a GeneAmp PCR System 9600 PCR machine (Perkin Elmer), utilizing a touch-down PCR protocol. The protocol, unless indicated otherwise in Table 3, consisted of an initial incubation of 95° C. for 10 min, followed by eight cycles of 950C for 20 sec, 66° C. (minus 1° C. per cycle) for 15 sec, 72° C. for 2 min, and twenty-seven cycles of 95° C. for 20 sec, 54° C. for 15 sec, 72° C. for 2 min, and one final extension step of 72° C. for 10 min.

[0081] For the secondary PCR reactions, one microliter of each primary PCR reaction was re-amplified using the secondary PCR primers, also listed in Table 2. The thermal cycling profile that was used for the primary PCR for an exon was used for the secondary PCR. TABLE 3 Cycling Profile Modifications Exon Primary PCR Secondary PCR 1E Touch-Down PCR step: 8 cycles same as Primary PCR 64 C (minus 1 C per cycle), for 15 sec Total Number of cycles: 35 1F Touch-Down PCR step: 10 cycles same as Primary PCR 64 C (minus 1 C per cycle), for 15 sec Total Number of cycles: 35 1G Touch-Down PCR step: 7 cycles same as Primary PCR 64 C (minus 1 C per cycle), for 15 sec Total Number of cycles: 35 1H Touch-Down PCR step: 10 cycles same as Primary PCR 66 C (minus 1 C per cycle), for 15 sec Total Number of cycles: 35

[0082] DNA sequencing. PCR products from 48 individuals, approximately one-third representing each of the 3 major racial groups (see above), were prepared for sequencing by treating 8 μL of each PCR product with 0.15 μL of exonuclease I (1.5 U/reaction), 0.3 μL of Shrimp Alkaline Phosphatase (0.3 U/reaction), q.s. to 10 μL with MilliΩ water, and incubated at 37° C. for 15 min, followed by 72° C. for 15 min. Cycle sequencing was performed on the GeneAmp PCR System 9600 PCR machine (Perkin Elmer) using the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit according to the manufacturer's directions, with the following changes: (1) 2 μL of dRhodamine terminator premix, instead of 8 μL; and (2) 10% (v/v) Dimethylsulfoxide was added to each individual nucleotide. The oligonucleotide primers (unlabeled), at 3 picomoles per reaction, used for the sequencing reactions are listed in Table 4. Sequencing reactions, with a final volume of 5 μL, were subjected to 30 cycles at 96° C. for 20 sec, 50° C. for 5 sec, and 60° C. for 4 min, followed by ethanol precipitation. After decanting the ethanol, samples were evaporated to dryness using a SpeedVac for roughly 15 min and were resuspended in 2 μL of loading buffer (5:1 deionized formamide:50 mM EDTA pH 8.0). The samples were then, heated to 94° C. for 2 min, and electrophoresed through 5.25% polyacrylamide/6M urea gels in an ABI Prism 377 DNA Sequencer according to the manufacturer's instructions for sequence determination. All sequences were determined from both the 5′ and 3′ (sense and antisense) direction.

[0083] Of the forty-eight samples, 38 polymorphisms were identified. The polymorphisms are described in Table 5 below. TABLE 4 Sequencing Primers (No. = Polymorphism No.) No. FORWARD PRIMER REVERSE PRIMER  1 CTCTGGCAGGAGCAAAG (SEQ ID NO:51) ACAGTGGGCAGAGACAG (SEQ ID NO:52)  2 GTGGTTTATTCCCCGTAT (SEQ ID NO:53) ATACACACCTGGGATAGTGG (SEQ ID NO:54)  3-5 GGTAATTAAGATGAAGAAAGCA (SEQ ID NO:55) GAAATGGCATAGGTTGTC (SEQ ID NO:56)  6 GGCCACACTCAACTGTA (SEQ ID NO:57) CTCAAAAAAAACACAGTAGG (SEQ ID NO:58)  7, 8 ACTTTTTCTGCCCCTTAT (SEQ ID NO:59) ATATGGAAGCACTTGTAAGTAAA (SEQ ID NO:60)  9-12 TTAAGACGAAGGAAACAATTCT (SEQ ID NO:61) AATGGCATACGTTGTCA (SEQ ID NO:62) 13, 14 AGAATGGCAATTATGAACA (SEQ ID NO:63) TGTGTGCCCTTAAAGTCT (SEQ ID NO:64) 15-17 AGAATGGCAATTATGAACA (SEQ ID NO:65) ACCTGAGATAGTGGCTTCC (SEQ ID NO:66) 18-24 CTCTGGC T  CTGTCCTAC* (SEQ ID NO:67) ACCTGAGATAGTGGCTTCC (SEQ ID NO:68) 25 ATCAAAGGGTAAAATTCAGA (SEQ ID NO:69) CAGCAGCTTGTCACCTAC (SEQ ID NO:70) 26 AATTTGCTTTTGAAAGAATC (SEQ ID NO:71) GGTAGGCCCAAATACTCA (SEQ ID NO:72) 27, 28 AATTTGCTTTTGAAAGAATC (SEQ ID NO:73) GGCAGTCCAAAAGAAATA (SEQ ID NO:74) 29, 30 TTTTGAGGGCAGGTTCTA (SEQ ID NO:75) CACCTCTGGCATGACTAC (SEQ ID NO:76) 31, 32 TTGCAGGAGTTTGTTTAAT (SEQ ID NO:77) AATGGGACAAATGTAAATGATA (SEQ ID NO:78) 33 CATTGCAGGAGTTTGTTTA (SEQ ID NO:79) CATCTGAGAACCCTAAGAGA (SEQ ID NO:80) 34 AGAAATAGCCTCTGAAATTC (SEQ ID NO:81) ATGTCAAATCACAATTCAGTAAGG (SEQ ID NO:82) 35 CCGCCTACTGTATCATAGCA (SEQ ID NO:83) GAGTGTACGAGGTTGAGTAAG (SEQ ID NO:84) 36-38 ATTTTGCCAGTATCTTTTTAG (SEQ ID NO:85) CAACGAAATGTCAAATCACAG (SEQ ID NO:86)

[0084] TABLE 5 UGT1 polymorphisms. Amino acid changes numbered from first methionine for that exon (Ex). No Ex Ntd AA SEQUENCE (SEQ ID NO:)  1 1A G 227 A Gly 71 Arg CATCAGAGAC A GAGCATTTTACACCTT (SEQ ID NO:87)  2 1A T 765 C Ser 251 Pro GGACCTATTGAGC C  CTGCATCTGTCT (SEQ ID NO:88)  3 1C T 75 C Trp 11 Arg GGTTCCCCTGCCG C  GGCTGGCCACA (SEQ ID NO:89)  4 1C G 125 A GCCCTGGGCTGA A  AGTGGAAAG (SEQ ID NO:90)  5 1C T 184 C Val 47 Ala ATGCGGGAGG C  CTTGCGGGAGCT (SEQ ID NO:91)  6 1C A 521 G CTCTGCGCGGC G  GTGCTGGCTAAG (SEQ ID NO:92)  7 1D G 848 A TACCCCAGGCC A  ATCATGCCCAACA (SEQ ID NO:93)  8 1D C 43 T Intronic TCCAGGCAAAA T  ACTTTTTAAAAAATG (SEQ ID NO:94)  9 1E T 187 C Leu 48 Ser AGCATGCGGGAGGCCT C  GCGGGA (SEQ ID NO:95) 10 1E C 194 G Asp 58 Glu GCGGGA G  CTCCATGCGAGAGG (SEQ ID NO:96) 11 1E T 232 C Leu 63 Pro TGGTGGTCCTCACCC C  GGAGGTGAA (SEQ ID NO:97) 12 1E A 257 G TACATCAAAGA G  GAGAACTTTTTCAC (SEQ ID NO:98) 13 1E C 468 A His 142 Asn TGATCAGGCACCTG A  ATGCTACTTCC (SEQ ID NO:99) 14 1E C 517 G Ala 158 Gly ACCTCTGCG G  GGCGGTGCTGG (SEQ ID NO:100) 15 1E C 689 T AAGAACATGCT T  TACCCTCTGGC (SEQ ID NO:101) 16 1E C 701 T CTCTGGC T  CTGTCCTACC (SEQ ID NO:102) 17 1E C 717 T TCCTACCTTTGC T  ATGCTGTTTCT (SEQ ID NO:103) 18 1E C 786 A Leu 248 Ile TGTCAGTGGTGGAT A  TT (SEQ ID NO:104) 19 1E G 789 C Val 249 Leu GGTGGAT A  TT C*  TCAGC (SEQ ID NO:105) 20 1E C 795 T His 251 Tyr TCAGC T  ATGCATC (SEQ ID NO:106) 21 1E T 803 C Ser 253 Phe GCATC C  GTGTGGCTGTTCCGA (SEQ ID NO:107) 22 1E G 819 C Gly 259 Arg TGGCTGTTCCGA C  GGGACTT (SEQ ID NO:108) 23 1E T 827 C GGGACTT C  GTGATGGA (SEQ ID NO:109) 24 1E T 836 C GTGATGGA C  TACCCCAGGCCGAT (SEQ ID NO:110) 25 1F T 161 G Ser 7 Ala CCTGCCTCCTTCGC G  CATTTCAGAG (SEQ ID NO:111) 26 1F A 457 G GCGATCATTCCT G  ACTGCTCCTCAG (SEQ ID NO:112) 27 1F A 683 G Thr 181 Ala CCCTGGAGCAT G  CATTCAGCAG (SEQ ID NO:113) 28 1F A 694 C Arg 184 Ser CATTCAGCAG C  AGCCCAGACCCT (SEQ ID NO:114) 29 1G T 35 G TACTTCTTCCAC G  TACTATATTA (SEQ ID NO:115) 30 1G C 124 A GGCCTCCTTCC A  CTATATGTGTGT (SEQ ID NO:116) 31 1G T 712 C Trp 208 Arg GGAGAGAGTA C  GGAACCACAT (SEQ ID NO:117) 32 1G G 846 A TCAATTTGGTT A  TTGCGAACTGA (SEQ ID NO:118) 33 1H G 518 C Gly 173 Ala CAGGGGAATAG C  TTGCCACTAT (SEQ ID NO:119) 34 1H A 765 G TGTTGCGAAC G  GACTTTGTTTTGG (SEQ ID NO:120) 35 1J G 127 A TTCACCAGCA A  TCGGTGGTGG (SEQ ID NO:121) 36 1J C 694 T CTAGAAATAGC T  TCTGAAATTCTCC (SEQ ID NO:122) 37 1J C 731 A Leu 244 Ile CGGCATATGAT A  TCTACAGTCACA (SEQ ID NO:123) 38 1J T 761 C Arg 254 Stop TCAATTTGGTTG C  TGCGAACAGGAC (SEQ ID NO:124)

[0085] The asterisk associated with the second nucleotide residue in polymorphism no. 19 is in the sequence surrounding the newly discovered polymorphism at residue 789 (nucleotide change from C at residue 786 to A).

[0086] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0087] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

1 124 1 864 DNA Homo sapiens CDS (1)...(864) 1 atg gct gtg gag tcc cag ggc gga cgc cca ctt gtc ctg ggc ctg ctg 48 Met Ala Val Glu Ser Gln Gly Gly Arg Pro Leu Val Leu Gly Leu Leu 1 5 10 15 ctg tgt gtg ctg ggc cca gtg gtg tcc cat gct ggg aag ata ctg ttg 96 Leu Cys Val Leu Gly Pro Val Val Ser His Ala Gly Lys Ile Leu Leu 20 25 30 atc cca gtg gat ggc agc cac tgg ctg agc atg ctt ggg gcc atc cag 144 Ile Pro Val Asp Gly Ser His Trp Leu Ser Met Leu Gly Ala Ile Gln 35 40 45 cag ctg cag cag agg gga cat gaa ata gtt gtc cta gca cct gac gcc 192 Gln Leu Gln Gln Arg Gly His Glu Ile Val Val Leu Ala Pro Asp Ala 50 55 60 tcg ttg tac atc aga gac gga gca ttt tac acc ttg aag acg tac cct 240 Ser Leu Tyr Ile Arg Asp Gly Ala Phe Tyr Thr Leu Lys Thr Tyr Pro 65 70 75 80 gtg cca ttc caa agg gag gat gtg aaa gag tct ttt gtt agt ctc ggg 288 Val Pro Phe Gln Arg Glu Asp Val Lys Glu Ser Phe Val Ser Leu Gly 85 90 95 cat aat gtt ttt gag aat gat tct ttc ctg cag cgt gtg atc aaa aca 336 His Asn Val Phe Glu Asn Asp Ser Phe Leu Gln Arg Val Ile Lys Thr 100 105 110 tac aag aaa ata aaa aag gac tct gct atg ctt ttg tct ggc tgt tcc 384 Tyr Lys Lys Ile Lys Lys Asp Ser Ala Met Leu Leu Ser Gly Cys Ser 115 120 125 cac tta ctg cac aac aag gag ctc atg gcc tcc ctg gca gaa agc agc 432 His Leu Leu His Asn Lys Glu Leu Met Ala Ser Leu Ala Glu Ser Ser 130 135 140 ttt gat gtc atg ctg acg gac cct ttc ctt cct tgc agc ccc atc gtg 480 Phe Asp Val Met Leu Thr Asp Pro Phe Leu Pro Cys Ser Pro Ile Val 145 150 155 160 gcc cag tac ctg tct ctg ccc act gta ttc ttc ttg cat gca ctg cca 528 Ala Gln Tyr Leu Ser Leu Pro Thr Val Phe Phe Leu His Ala Leu Pro 165 170 175 tgc agc ctg gaa ttt gag gct acc cag tgc ccc aac cca ttc tcc tac 576 Cys Ser Leu Glu Phe Glu Ala Thr Gln Cys Pro Asn Pro Phe Ser Tyr 180 185 190 gtg ccc agg cct ctc tcc tct cat tca gat cac atg acc ttc ctg cag 624 Val Pro Arg Pro Leu Ser Ser His Ser Asp His Met Thr Phe Leu Gln 195 200 205 cgg gtg aag aac atg ctc att gcc ttt tca cag aac ttt ctg tgc gac 672 Arg Val Lys Asn Met Leu Ile Ala Phe Ser Gln Asn Phe Leu Cys Asp 210 215 220 gtg gtt tat tcc ccg tat gca acc ctt gcc tca gaa ttc ctt cag aga 720 Val Val Tyr Ser Pro Tyr Ala Thr Leu Ala Ser Glu Phe Leu Gln Arg 225 230 235 240 gag gtg act gtc cag gac cta ttg agc tct gca tct gtc tgg ctg ttt 768 Glu Val Thr Val Gln Asp Leu Leu Ser Ser Ala Ser Val Trp Leu Phe 245 250 255 aga agt gac ttt gtg aag gat tac cct agg ccc atc atg ccc aat atg 816 Arg Ser Asp Phe Val Lys Asp Tyr Pro Arg Pro Ile Met Pro Asn Met 260 265 270 gtt ttt gtt ggt gga atc aac tgc ctt cac caa aat cca cta tcc cag 864 Val Phe Val Gly Gly Ile Asn Cys Leu His Gln Asn Pro Leu Ser Gln 275 280 285 2 288 PRT Homo sapiens 2 Met Ala Val Glu Ser Gln Gly Gly Arg Pro Leu Val Leu Gly Leu Leu 1 5 10 15 Leu Cys Val Leu Gly Pro Val Val Ser His Ala Gly Lys Ile Leu Leu 20 25 30 Ile Pro Val Asp Gly Ser His Trp Leu Ser Met Leu Gly Ala Ile Gln 35 40 45 Gln Leu Gln Gln Arg Gly His Glu Ile Val Val Leu Ala Pro Asp Ala 50 55 60 Ser Leu Tyr Ile Arg Asp Gly Ala Phe Tyr Thr Leu Lys Thr Tyr Pro 65 70 75 80 Val Pro Phe Gln Arg Glu Asp Val Lys Glu Ser Phe Val Ser Leu Gly 85 90 95 His Asn Val Phe Glu Asn Asp Ser Phe Leu Gln Arg Val Ile Lys Thr 100 105 110 Tyr Lys Lys Ile Lys Lys Asp Ser Ala Met Leu Leu Ser Gly Cys Ser 115 120 125 His Leu Leu His Asn Lys Glu Leu Met Ala Ser Leu Ala Glu Ser Ser 130 135 140 Phe Asp Val Met Leu Thr Asp Pro Phe Leu Pro Cys Ser Pro Ile Val 145 150 155 160 Ala Gln Tyr Leu Ser Leu Pro Thr Val Phe Phe Leu His Ala Leu Pro 165 170 175 Cys Ser Leu Glu Phe Glu Ala Thr Gln Cys Pro Asn Pro Phe Ser Tyr 180 185 190 Val Pro Arg Pro Leu Ser Ser His Ser Asp His Met Thr Phe Leu Gln 195 200 205 Arg Val Lys Asn Met Leu Ile Ala Phe Ser Gln Asn Phe Leu Cys Asp 210 215 220 Val Val Tyr Ser Pro Tyr Ala Thr Leu Ala Ser Glu Phe Leu Gln Arg 225 230 235 240 Glu Val Thr Val Gln Asp Leu Leu Ser Ser Ala Ser Val Trp Leu Phe 245 250 255 Arg Ser Asp Phe Val Lys Asp Tyr Pro Arg Pro Ile Met Pro Asn Met 260 265 270 Val Phe Val Gly Gly Ile Asn Cys Leu His Gln Asn Pro Leu Ser Gln 275 280 285 3 867 DNA Homo sapiens CDS (1)...(867) 3 atg gcc aca gga ctc cag gtt ccc ctg ccg tgg ctg gcc aca gga ctg 48 Met Ala Thr Gly Leu Gln Val Pro Leu Pro Trp Leu Ala Thr Gly Leu 1 5 10 15 ctg ctt ctc ctc agt gtc cag ccc tgg gct gag agt gga aag gtg ttg 96 Leu Leu Leu Leu Ser Val Gln Pro Trp Ala Glu Ser Gly Lys Val Leu 20 25 30 gtg gtg ccc att gat ggc agc cac tgg ctc agc atg cgg gag gtc ttg 144 Val Val Pro Ile Asp Gly Ser His Trp Leu Ser Met Arg Glu Val Leu 35 40 45 cgg gag ctc cat gcc aga ggc cac cag gca gtg gtc ctc acc cca gag 192 Arg Glu Leu His Ala Arg Gly His Gln Ala Val Val Leu Thr Pro Glu 50 55 60 gtg aat atg cac atc aaa gaa gag aac ttt ttc acc ctg aca acc tat 240 Val Asn Met His Ile Lys Glu Glu Asn Phe Phe Thr Leu Thr Thr Tyr 65 70 75 80 gcc att tcg tgg acc cag gat gaa ttt gat cgc cat gtg ctg ggc cac 288 Ala Ile Ser Trp Thr Gln Asp Glu Phe Asp Arg His Val Leu Gly His 85 90 95 act caa ctg tac ttt gaa aca gaa cat ttt ctg aag aaa ttt ttc aga 336 Thr Gln Leu Tyr Phe Glu Thr Glu His Phe Leu Lys Lys Phe Phe Arg 100 105 110 agt atg gca atg ttg aac aat atg tct ttg gtc tat cat agg tct tgt 384 Ser Met Ala Met Leu Asn Asn Met Ser Leu Val Tyr His Arg Ser Cys 115 120 125 gtg gag cta cta cat aat gag gcc ctg atc agg cac ctg aat gct act 432 Val Glu Leu Leu His Asn Glu Ala Leu Ile Arg His Leu Asn Ala Thr 130 135 140 tcc ttt gat gtg gtt tta aca gac ccc gtt aac ctc tgc gcg gca gtg 480 Ser Phe Asp Val Val Leu Thr Asp Pro Val Asn Leu Cys Ala Ala Val 145 150 155 160 ctg gct aag tac ctg tcg att cct act gtg ttt ttt ttg agg aac att 528 Leu Ala Lys Tyr Leu Ser Ile Pro Thr Val Phe Phe Leu Arg Asn Ile 165 170 175 cca tgt gat tta gac ttt aag ggc aca cag tgt cca aac cct tcc tcc 576 Pro Cys Asp Leu Asp Phe Lys Gly Thr Gln Cys Pro Asn Pro Ser Ser 180 185 190 tat att cct aga tta cta aca acc aat tca gac cac atg aca ttc atg 624 Tyr Ile Pro Arg Leu Leu Thr Thr Asn Ser Asp His Met Thr Phe Met 195 200 205 caa agg gtc aag aac atg ctc tac cct ctg gcc ctg tcc tac att tgc 672 Gln Arg Val Lys Asn Met Leu Tyr Pro Leu Ala Leu Ser Tyr Ile Cys 210 215 220 cat gct ttt tct gct cct tat gca agc ctt gcc tct gag ctt ttt cag 720 His Ala Phe Ser Ala Pro Tyr Ala Ser Leu Ala Ser Glu Leu Phe Gln 225 230 235 240 aga gag gtg tca gtg gtg gat att ctc agt cat gca tct gtg tgg ctg 768 Arg Glu Val Ser Val Val Asp Ile Leu Ser His Ala Ser Val Trp Leu 245 250 255 ttc cga ggg gac ttt gtg atg gac tac ccc agg cca atc atg ccc aac 816 Phe Arg Gly Asp Phe Val Met Asp Tyr Pro Arg Pro Ile Met Pro Asn 260 265 270 atg gtc ttc att ggg ggc atc aac tgt gcc aac agg aag cca cta tct 864 Met Val Phe Ile Gly Gly Ile Asn Cys Ala Asn Arg Lys Pro Leu Ser 275 280 285 cag 867 Gln 4 289 PRT Homo sapiens 4 Met Ala Thr Gly Leu Gln Val Pro Leu Pro Trp Leu Ala Thr Gly Leu 1 5 10 15 Leu Leu Leu Leu Ser Val Gln Pro Trp Ala Glu Ser Gly Lys Val Leu 20 25 30 Val Val Pro Ile Asp Gly Ser His Trp Leu Ser Met Arg Glu Val Leu 35 40 45 Arg Glu Leu His Ala Arg Gly His Gln Ala Val Val Leu Thr Pro Glu 50 55 60 Val Asn Met His Ile Lys Glu Glu Asn Phe Phe Thr Leu Thr Thr Tyr 65 70 75 80 Ala Ile Ser Trp Thr Gln Asp Glu Phe Asp Arg His Val Leu Gly His 85 90 95 Thr Gln Leu Tyr Phe Glu Thr Glu His Phe Leu Lys Lys Phe Phe Arg 100 105 110 Ser Met Ala Met Leu Asn Asn Met Ser Leu Val Tyr His Arg Ser Cys 115 120 125 Val Glu Leu Leu His Asn Glu Ala Leu Ile Arg His Leu Asn Ala Thr 130 135 140 Ser Phe Asp Val Val Leu Thr Asp Pro Val Asn Leu Cys Ala Ala Val 145 150 155 160 Leu Ala Lys Tyr Leu Ser Ile Pro Thr Val Phe Phe Leu Arg Asn Ile 165 170 175 Pro Cys Asp Leu Asp Phe Lys Gly Thr Gln Cys Pro Asn Pro Ser Ser 180 185 190 Tyr Ile Pro Arg Leu Leu Thr Thr Asn Ser Asp His Met Thr Phe Met 195 200 205 Gln Arg Val Lys Asn Met Leu Tyr Pro Leu Ala Leu Ser Tyr Ile Cys 210 215 220 His Ala Phe Ser Ala Pro Tyr Ala Ser Leu Ala Ser Glu Leu Phe Gln 225 230 235 240 Arg Glu Val Ser Val Val Asp Ile Leu Ser His Ala Ser Val Trp Leu 245 250 255 Phe Arg Gly Asp Phe Val Met Asp Tyr Pro Arg Pro Ile Met Pro Asn 260 265 270 Met Val Phe Ile Gly Gly Ile Asn Cys Ala Asn Arg Lys Pro Leu Ser 275 280 285 Gln 5 867 DNA Homo sapiens CDS (1)...(867) 5 atg gcc aga gga ctc cag gtt ccc ctg ccg cgg ctg gcc aca gga ctg 48 Met Ala Arg Gly Leu Gln Val Pro Leu Pro Arg Leu Ala Thr Gly Leu 1 5 10 15 ctg ctc ctc ctc agt gtc cag ccc tgg gct gag agt gga aag gtg ttg 96 Leu Leu Leu Leu Ser Val Gln Pro Trp Ala Glu Ser Gly Lys Val Leu 20 25 30 gtg gtg ccc act gat ggc agc ccc tgg ctc agc atg cgg gag gcc ttg 144 Val Val Pro Thr Asp Gly Ser Pro Trp Leu Ser Met Arg Glu Ala Leu 35 40 45 cgg gag ctc cat gcc aga ggc cac cag gcg gtg gtc ctc acc cca gag 192 Arg Glu Leu His Ala Arg Gly His Gln Ala Val Val Leu Thr Pro Glu 50 55 60 gtg aat atg cac atc aaa gaa gag aaa ttt ttc acc ctg aca gcc tat 240 Val Asn Met His Ile Lys Glu Glu Lys Phe Phe Thr Leu Thr Ala Tyr 65 70 75 80 gct gtt cca tgg acc cag aag gaa ttt gat cgc gtt acg ctg ggc tac 288 Ala Val Pro Trp Thr Gln Lys Glu Phe Asp Arg Val Thr Leu Gly Tyr 85 90 95 act caa ggg ttc ttt gaa aca gaa cat ctt ctg aag aga tat tct aga 336 Thr Gln Gly Phe Phe Glu Thr Glu His Leu Leu Lys Arg Tyr Ser Arg 100 105 110 agt atg gca att atg aac aat gta tct ttg gcc ctt cat agg tgt tgt 384 Ser Met Ala Ile Met Asn Asn Val Ser Leu Ala Leu His Arg Cys Cys 115 120 125 gtg gag cta ctg cat aat gag gcc ctg atc agg cac ctg aat gct act 432 Val Glu Leu Leu His Asn Glu Ala Leu Ile Arg His Leu Asn Ala Thr 130 135 140 tcc ttt gat gtg gtt tta aca gac ccc gtt aac ctc tgt ggg gcg gtg 480 Ser Phe Asp Val Val Leu Thr Asp Pro Val Asn Leu Cys Gly Ala Val 145 150 155 160 ctg gct aag tac ctg tcg att cct gct gtg ttt ttt tgg agg tac att 528 Leu Ala Lys Tyr Leu Ser Ile Pro Ala Val Phe Phe Trp Arg Tyr Ile 165 170 175 cca tgt gac tta gac ttt aag ggc aca cag tgt cca aat cct tcc tcc 576 Pro Cys Asp Leu Asp Phe Lys Gly Thr Gln Cys Pro Asn Pro Ser Ser 180 185 190 tat att cct aag tta cta acg acc aat tca gac cac atg aca ttc ctg 624 Tyr Ile Pro Lys Leu Leu Thr Thr Asn Ser Asp His Met Thr Phe Leu 195 200 205 caa agg gtc aag aac atg ctc tac cct ctg gcc ctg tcc tac att tgc 672 Gln Arg Val Lys Asn Met Leu Tyr Pro Leu Ala Leu Ser Tyr Ile Cys 210 215 220 cat act ttt tct gcc cct tat gca agt ctt gcc tct gag ctt ttt cag 720 His Thr Phe Ser Ala Pro Tyr Ala Ser Leu Ala Ser Glu Leu Phe Gln 225 230 235 240 aga gag gtg tca gtg gtg gat ctt gtc agc tat gca tcc gtg tgg ctg 768 Arg Glu Val Ser Val Val Asp Leu Val Ser Tyr Ala Ser Val Trp Leu 245 250 255 ttc cga ggg gac ttt gtg atg gac tac ccc agg ccg atc atg ccc aac 816 Phe Arg Gly Asp Phe Val Met Asp Tyr Pro Arg Pro Ile Met Pro Asn 260 265 270 atg gtc ttc att ggg ggc atc aac tgt gcc aac ggg aag cca cta tct 864 Met Val Phe Ile Gly Gly Ile Asn Cys Ala Asn Gly Lys Pro Leu Ser 275 280 285 cag 867 Gln 6 289 PRT Homo sapiens 6 Met Ala Arg Gly Leu Gln Val Pro Leu Pro Arg Leu Ala Thr Gly Leu 1 5 10 15 Leu Leu Leu Leu Ser Val Gln Pro Trp Ala Glu Ser Gly Lys Val Leu 20 25 30 Val Val Pro Thr Asp Gly Ser Pro Trp Leu Ser Met Arg Glu Ala Leu 35 40 45 Arg Glu Leu His Ala Arg Gly His Gln Ala Val Val Leu Thr Pro Glu 50 55 60 Val Asn Met His Ile Lys Glu Glu Lys Phe Phe Thr Leu Thr Ala Tyr 65 70 75 80 Ala Val Pro Trp Thr Gln Lys Glu Phe Asp Arg Val Thr Leu Gly Tyr 85 90 95 Thr Gln Gly Phe Phe Glu Thr Glu His Leu Leu Lys Arg Tyr Ser Arg 100 105 110 Ser Met Ala Ile Met Asn Asn Val Ser Leu Ala Leu His Arg Cys Cys 115 120 125 Val Glu Leu Leu His Asn Glu Ala Leu Ile Arg His Leu Asn Ala Thr 130 135 140 Ser Phe Asp Val Val Leu Thr Asp Pro Val Asn Leu Cys Gly Ala Val 145 150 155 160 Leu Ala Lys Tyr Leu Ser Ile Pro Ala Val Phe Phe Trp Arg Tyr Ile 165 170 175 Pro Cys Asp Leu Asp Phe Lys Gly Thr Gln Cys Pro Asn Pro Ser Ser 180 185 190 Tyr Ile Pro Lys Leu Leu Thr Thr Asn Ser Asp His Met Thr Phe Leu 195 200 205 Gln Arg Val Lys Asn Met Leu Tyr Pro Leu Ala Leu Ser Tyr Ile Cys 210 215 220 His Thr Phe Ser Ala Pro Tyr Ala Ser Leu Ala Ser Glu Leu Phe Gln 225 230 235 240 Arg Glu Val Ser Val Val Asp Leu Val Ser Tyr Ala Ser Val Trp Leu 245 250 255 Phe Arg Gly Asp Phe Val Met Asp Tyr Pro Arg Pro Ile Met Pro Asn 260 265 270 Met Val Phe Ile Gly Gly Ile Asn Cys Ala Asn Gly Lys Pro Leu Ser 275 280 285 Gln 7 867 DNA Homo sapiens CDS (1)...(867) 7 atg gcc aca gga ctc cag gtt ccc ctg ccg cag ctg gcc aca gga ctg 48 Met Ala Thr Gly Leu Gln Val Pro Leu Pro Gln Leu Ala Thr Gly Leu 1 5 10 15 ctg ctt ctc ctc agt gtc cag ccc tgg gct gag agt ggg aag gtg ctg 96 Leu Leu Leu Leu Ser Val Gln Pro Trp Ala Glu Ser Gly Lys Val Leu 20 25 30 gtg gtg ccc act gat ggc agc cac tgg ctc agc atg cgg gag gcc ttg 144 Val Val Pro Thr Asp Gly Ser His Trp Leu Ser Met Arg Glu Ala Leu 35 40 45 cgg gac ctc cat gcg aga ggc cac cag gtg gtg gtc ctc acc ctg gag 192 Arg Asp Leu His Ala Arg Gly His Gln Val Val Val Leu Thr Leu Glu 50 55 60 gtg aat atg tac atc aaa gaa gag aac ttt ttc acc ctg aca acg tat 240 Val Asn Met Tyr Ile Lys Glu Glu Asn Phe Phe Thr Leu Thr Thr Tyr 65 70 75 80 gcc att tca tgg acc cag gac gaa ttt gat cgc ctt ttg ctg ggt cac 288 Ala Ile Ser Trp Thr Gln Asp Glu Phe Asp Arg Leu Leu Leu Gly His 85 90 95 act caa tcg ttc ttt gaa aca gaa cat ctt ctg atg aaa ttt tct aga 336 Thr Gln Ser Phe Phe Glu Thr Glu His Leu Leu Met Lys Phe Ser Arg 100 105 110 aga atg gca att atg aac aat atg tct ttg atc ata cat agg tct tgt 384 Arg Met Ala Ile Met Asn Asn Met Ser Leu Ile Ile His Arg Ser Cys 115 120 125 gtg gag cta ctg cat aat gag gcc ctg atc agg cac ctg cat gct act 432 Val Glu Leu Leu His Asn Glu Ala Leu Ile Arg His Leu His Ala Thr 130 135 140 tcc ttt gat gtg gtt cta aca gac ccc ttt cac ctc tgc gcg gcg gtg 480 Ser Phe Asp Val Val Leu Thr Asp Pro Phe His Leu Cys Ala Ala Val 145 150 155 160 ctg gct aag tac ctg tcg att cct gct gtg ttt ttc ttg agg aac att 528 Leu Ala Lys Tyr Leu Ser Ile Pro Ala Val Phe Phe Leu Arg Asn Ile 165 170 175 cca tgt gat tta gac ttt aag ggc aca cag tgt cca aac cct tcc tcc 576 Pro Cys Asp Leu Asp Phe Lys Gly Thr Gln Cys Pro Asn Pro Ser Ser 180 185 190 tat att cct aga tta cta acg acc aat tca gac cac atg aca ttc ctg 624 Tyr Ile Pro Arg Leu Leu Thr Thr Asn Ser Asp His Met Thr Phe Leu 195 200 205 caa agg gtc aag aac atg ctc tac cct ctg gcc ctg tcc tac ctt tgc 672 Gln Arg Val Lys Asn Met Leu Tyr Pro Leu Ala Leu Ser Tyr Leu Cys 210 215 220 cat gct gtt tct gct cct tat gca agc ctt gcc tct gag ctt ttt cag 720 His Ala Val Ser Ala Pro Tyr Ala Ser Leu Ala Ser Glu Leu Phe Gln 225 230 235 240 aga gag gtg tca gtg gtg gat ctt gtc agc cat gca tct gtg tgg ctg 768 Arg Glu Val Ser Val Val Asp Leu Val Ser His Ala Ser Val Trp Leu 245 250 255 ttc cga ggg gac ttt gtg atg gat tac ccc agg ccg atc atg ccc aac 816 Phe Arg Gly Asp Phe Val Met Asp Tyr Pro Arg Pro Ile Met Pro Asn 260 265 270 atg gtc ttc att ggg ggc atc aac tgt gcc aac ggg aag cca cta tct 864 Met Val Phe Ile Gly Gly Ile Asn Cys Ala Asn Gly Lys Pro Leu Ser 275 280 285 cag 867 Gln 8 289 PRT Homo sapiens 8 Met Ala Thr Gly Leu Gln Val Pro Leu Pro Gln Leu Ala Thr Gly Leu 1 5 10 15 Leu Leu Leu Leu Ser Val Gln Pro Trp Ala Glu Ser Gly Lys Val Leu 20 25 30 Val Val Pro Thr Asp Gly Ser His Trp Leu Ser Met Arg Glu Ala Leu 35 40 45 Arg Asp Leu His Ala Arg Gly His Gln Val Val Val Leu Thr Leu Glu 50 55 60 Val Asn Met Tyr Ile Lys Glu Glu Asn Phe Phe Thr Leu Thr Thr Tyr 65 70 75 80 Ala Ile Ser Trp Thr Gln Asp Glu Phe Asp Arg Leu Leu Leu Gly His 85 90 95 Thr Gln Ser Phe Phe Glu Thr Glu His Leu Leu Met Lys Phe Ser Arg 100 105 110 Arg Met Ala Ile Met Asn Asn Met Ser Leu Ile Ile His Arg Ser Cys 115 120 125 Val Glu Leu Leu His Asn Glu Ala Leu Ile Arg His Leu His Ala Thr 130 135 140 Ser Phe Asp Val Val Leu Thr Asp Pro Phe His Leu Cys Ala Ala Val 145 150 155 160 Leu Ala Lys Tyr Leu Ser Ile Pro Ala Val Phe Phe Leu Arg Asn Ile 165 170 175 Pro Cys Asp Leu Asp Phe Lys Gly Thr Gln Cys Pro Asn Pro Ser Ser 180 185 190 Tyr Ile Pro Arg Leu Leu Thr Thr Asn Ser Asp His Met Thr Phe Leu 195 200 205 Gln Arg Val Lys Asn Met Leu Tyr Pro Leu Ala Leu Ser Tyr Leu Cys 210 215 220 His Ala Val Ser Ala Pro Tyr Ala Ser Leu Ala Ser Glu Leu Phe Gln 225 230 235 240 Arg Glu Val Ser Val Val Asp Leu Val Ser His Ala Ser Val Trp Leu 245 250 255 Phe Arg Gly Asp Phe Val Met Asp Tyr Pro Arg Pro Ile Met Pro Asn 260 265 270 Met Val Phe Ile Gly Gly Ile Asn Cys Ala Asn Gly Lys Pro Leu Ser 275 280 285 Gln 9 861 DNA Homo sapiens CDS (1)...(861) 9 atg gcc tgc ctc ctt cgc tca ttt cag aga att tct gca ggg gtt ttc 48 Met Ala Cys Leu Leu Arg Ser Phe Gln Arg Ile Ser Ala Gly Val Phe 1 5 10 15 ttc tta gca ctt tgg ggc atg gtt gta ggt gac aag ctg ctg gtg gtc 96 Phe Leu Ala Leu Trp Gly Met Val Val Gly Asp Lys Leu Leu Val Val 20 25 30 cct cag gac gga agc cac tgg ctt agt atg aag gat ata gtt gag gtt 144 Pro Gln Asp Gly Ser His Trp Leu Ser Met Lys Asp Ile Val Glu Val 35 40 45 ctc agt gac cgg ggt cat gag att gta gtg gtg gtg cct gaa gtt aat 192 Leu Ser Asp Arg Gly His Glu Ile Val Val Val Val Pro Glu Val Asn 50 55 60 ttg ctt ttg aaa gaa tcc aaa tac tac aca aga aaa atc tat cca gtg 240 Leu Leu Leu Lys Glu Ser Lys Tyr Tyr Thr Arg Lys Ile Tyr Pro Val 65 70 75 80 ccg tat gac caa gaa gag ctg aag aac cgt tac caa tca ttt gga aac 288 Pro Tyr Asp Gln Glu Glu Leu Lys Asn Arg Tyr Gln Ser Phe Gly Asn 85 90 95 aat cac ttt gct gag cga tca ttc cta act gct cct cag aca gag tac 336 Asn His Phe Ala Glu Arg Ser Phe Leu Thr Ala Pro Gln Thr Glu Tyr 100 105 110 agg aat aac atg att gtt att ggc ctg tac ttc atc aac tgc cag agc 384 Arg Asn Asn Met Ile Val Ile Gly Leu Tyr Phe Ile Asn Cys Gln Ser 115 120 125 ctc ctg cag gac agg gac acc ctg aac ttc ttt aag gag agc aag ttt 432 Leu Leu Gln Asp Arg Asp Thr Leu Asn Phe Phe Lys Glu Ser Lys Phe 130 135 140 gat gct ctt ttc aca gac cca gcc tta ccc tgt ggg gtg atc ctg gct 480 Asp Ala Leu Phe Thr Asp Pro Ala Leu Pro Cys Gly Val Ile Leu Ala 145 150 155 160 gag tat ttg ggc cta cca tct gtg tac ctc ttc agg ggt ttt ccg tgt 528 Glu Tyr Leu Gly Leu Pro Ser Val Tyr Leu Phe Arg Gly Phe Pro Cys 165 170 175 tcc ctg gag cat aca ttc agc aga agc cca gac cct gtg tcc tac att 576 Ser Leu Glu His Thr Phe Ser Arg Ser Pro Asp Pro Val Ser Tyr Ile 180 185 190 ccc agg tgc tac aca aag ttt tca gac cac atg act ttt tcc caa cga 624 Pro Arg Cys Tyr Thr Lys Phe Ser Asp His Met Thr Phe Ser Gln Arg 195 200 205 gtg gcc aac ttc ctt gtt aat ttg ttg gag ccc tat cta ttt tat tgt 672 Val Ala Asn Phe Leu Val Asn Leu Leu Glu Pro Tyr Leu Phe Tyr Cys 210 215 220 ctg ttt tca aag tat gaa gaa ctc gca tca gct gtc ctc aag aga gat 720 Leu Phe Ser Lys Tyr Glu Glu Leu Ala Ser Ala Val Leu Lys Arg Asp 225 230 235 240 gtg gat ata atc acc tta tat cag aag gtc tct gtt tgg ctg tta aga 768 Val Asp Ile Ile Thr Leu Tyr Gln Lys Val Ser Val Trp Leu Leu Arg 245 250 255 tat gac ttt gtg ctt gaa tat cct agg ccg gtc atg ccc aac atg gtc 816 Tyr Asp Phe Val Leu Glu Tyr Pro Arg Pro Val Met Pro Asn Met Val 260 265 270 ttc att gga ggt atc aac tgt aag aag agg aaa gac ttg tct cag 861 Phe Ile Gly Gly Ile Asn Cys Lys Lys Arg Lys Asp Leu Ser Gln 275 280 285 10 287 PRT Homo sapiens 10 Met Ala Cys Leu Leu Arg Ser Phe Gln Arg Ile Ser Ala Gly Val Phe 1 5 10 15 Phe Leu Ala Leu Trp Gly Met Val Val Gly Asp Lys Leu Leu Val Val 20 25 30 Pro Gln Asp Gly Ser His Trp Leu Ser Met Lys Asp Ile Val Glu Val 35 40 45 Leu Ser Asp Arg Gly His Glu Ile Val Val Val Val Pro Glu Val Asn 50 55 60 Leu Leu Leu Lys Glu Ser Lys Tyr Tyr Thr Arg Lys Ile Tyr Pro Val 65 70 75 80 Pro Tyr Asp Gln Glu Glu Leu Lys Asn Arg Tyr Gln Ser Phe Gly Asn 85 90 95 Asn His Phe Ala Glu Arg Ser Phe Leu Thr Ala Pro Gln Thr Glu Tyr 100 105 110 Arg Asn Asn Met Ile Val Ile Gly Leu Tyr Phe Ile Asn Cys Gln Ser 115 120 125 Leu Leu Gln Asp Arg Asp Thr Leu Asn Phe Phe Lys Glu Ser Lys Phe 130 135 140 Asp Ala Leu Phe Thr Asp Pro Ala Leu Pro Cys Gly Val Ile Leu Ala 145 150 155 160 Glu Tyr Leu Gly Leu Pro Ser Val Tyr Leu Phe Arg Gly Phe Pro Cys 165 170 175 Ser Leu Glu His Thr Phe Ser Arg Ser Pro Asp Pro Val Ser Tyr Ile 180 185 190 Pro Arg Cys Tyr Thr Lys Phe Ser Asp His Met Thr Phe Ser Gln Arg 195 200 205 Val Ala Asn Phe Leu Val Asn Leu Leu Glu Pro Tyr Leu Phe Tyr Cys 210 215 220 Leu Phe Ser Lys Tyr Glu Glu Leu Ala Ser Ala Val Leu Lys Arg Asp 225 230 235 240 Val Asp Ile Ile Thr Leu Tyr Gln Lys Val Ser Val Trp Leu Leu Arg 245 250 255 Tyr Asp Phe Val Leu Glu Tyr Pro Arg Pro Val Met Pro Asn Met Val 260 265 270 Phe Ile Gly Gly Ile Asn Cys Lys Lys Arg Lys Asp Leu Ser Gln 275 280 285 11 1115 DNA Homo sapiens CDS (165)...(1115) 11 tgta ttattatgag taaatcattg gcagtgaatg tgaatttttt 44 tttaaatgaa tgaataagta cacgccttct tttgagggca ggttctatct gtacttcttc 104 cacttactat attataggag cttagaatcc cagctgctgg ctctgggctg aagttctctg 164 atg gct cgt gca ggg tgg act ggc ctc ctt ccc cta tat gtg tgt cta 212 Met Ala Arg Ala Gly Trp Thr Gly Leu Leu Pro Leu Tyr Val Cys Leu 1 5 10 15 ctg ctg acc tgt gct ttg cca agg tca ggg aag ctg ctg gta gtg ccc 260 Leu Leu Thr Cys Ala Leu Pro Arg Ser Gly Lys Leu Leu Val Val Pro 20 25 30 atg gat ggg agc cac tgg ttc acc atg cag tcg gtg gtg gag aaa ctc 308 Met Asp Gly Ser His Trp Phe Thr Met Gln Ser Val Val Glu Lys Leu 35 40 45 atc ctc agg ggg cat gag gtg gtc gta gtc atg cca gag gtg agt tgg 356 Ile Leu Arg Gly His Glu Val Val Val Val Met Pro Glu Val Ser Trp 50 55 60 caa ctg gga aga tca ctg aat tgc aca gtg aag act tac tca acc tca 404 Gln Leu Gly Arg Ser Leu Asn Cys Thr Val Lys Thr Tyr Ser Thr Ser 65 70 75 80 tac act ctg gag gat cag gac cgg gag ttc atg gtt ttt gcc gat gct 452 Tyr Thr Leu Glu Asp Gln Asp Arg Glu Phe Met Val Phe Ala Asp Ala 85 90 95 cgc tgg acg gca cca ttg cga agt gca ttt tct cta tta aca agt tca 500 Arg Trp Thr Ala Pro Leu Arg Ser Ala Phe Ser Leu Leu Thr Ser Ser 100 105 110 tcc aat ggt att ttt gac tta ttt ttt tca aat tgc agg agt ttg ttt 548 Ser Asn Gly Ile Phe Asp Leu Phe Phe Ser Asn Cys Arg Ser Leu Phe 115 120 125 aat gac cga aaa tta gta gaa tac tta aag gag agt tgt ttt gat gca 596 Asn Asp Arg Lys Leu Val Glu Tyr Leu Lys Glu Ser Cys Phe Asp Ala 130 135 140 gtg ttt ctc gat cct ttt gat cgc tgt ggc tta att gtt gcc aaa tat 644 Val Phe Leu Asp Pro Phe Asp Arg Cys Gly Leu Ile Val Ala Lys Tyr 145 150 155 160 ttc tcc ctc ccc tct gtg gtc ttc gcc agg gga ata ttt tgc cac tat 692 Phe Ser Leu Pro Ser Val Val Phe Ala Arg Gly Ile Phe Cys His Tyr 165 170 175 ctt gaa gaa ggt gca cag tgc cct gct cct ctt tcc tat gtc ccc aga 740 Leu Glu Glu Gly Ala Gln Cys Pro Ala Pro Leu Ser Tyr Val Pro Arg 180 185 190 ctt ctc tta ggg ttc tca gac gcc atg act ttc aag gag aga gta tgg 788 Leu Leu Leu Gly Phe Ser Asp Ala Met Thr Phe Lys Glu Arg Val Trp 195 200 205 aac cac atc atg cac ttg gag gaa cat tta ttt tgc ccc tat ttt ttc 836 Asn His Ile Met His Leu Glu Glu His Leu Phe Cys Pro Tyr Phe Phe 210 215 220 aaa aat gtc tta gaa ata gcc tct gaa att ctc caa acc cct gtc acg 884 Lys Asn Val Leu Glu Ile Ala Ser Glu Ile Leu Gln Thr Pro Val Thr 225 230 235 240 gca tat gat ctc tac agc cac aca tca att tgg ttg ttg cga act gac 932 Ala Tyr Asp Leu Tyr Ser His Thr Ser Ile Trp Leu Leu Arg Thr Asp 245 250 255 ttt gtt ttg gag tat ccc aaa ccc gtg atg ccc aat atg atc ttc att 980 Phe Val Leu Glu Tyr Pro Lys Pro Val Met Pro Asn Met Ile Phe Ile 260 265 270 ggt ggt atc aac tgt cat cag gga aag cca gtg cct atg gta agt tat 1028 Gly Gly Ile Asn Cys His Gln Gly Lys Pro Val Pro Met Val Ser Tyr 275 280 285 ctc ccc ttt agc aca tta aga ata atc tgg ctt tgg aaa tta aaa gat 1076 Leu Pro Phe Ser Thr Leu Arg Ile Ile Trp Leu Trp Lys Leu Lys Asp 290 295 300 ttc tta cag aat cat aat tta tca ttt aca ttt gtc cca 1115 Phe Leu Gln Asn His Asn Leu Ser Phe Thr Phe Val Pro 305 310 315 12 317 PRT Homo sapiens 12 Met Ala Arg Ala Gly Trp Thr Gly Leu Leu Pro Leu Tyr Val Cys Leu 1 5 10 15 Leu Leu Thr Cys Ala Leu Pro Arg Ser Gly Lys Leu Leu Val Val Pro 20 25 30 Met Asp Gly Ser His Trp Phe Thr Met Gln Ser Val Val Glu Lys Leu 35 40 45 Ile Leu Arg Gly His Glu Val Val Val Val Met Pro Glu Val Ser Trp 50 55 60 Gln Leu Gly Arg Ser Leu Asn Cys Thr Val Lys Thr Tyr Ser Thr Ser 65 70 75 80 Tyr Thr Leu Glu Asp Gln Asp Arg Glu Phe Met Val Phe Ala Asp Ala 85 90 95 Arg Trp Thr Ala Pro Leu Arg Ser Ala Phe Ser Leu Leu Thr Ser Ser 100 105 110 Ser Asn Gly Ile Phe Asp Leu Phe Phe Ser Asn Cys Arg Ser Leu Phe 115 120 125 Asn Asp Arg Lys Leu Val Glu Tyr Leu Lys Glu Ser Cys Phe Asp Ala 130 135 140 Val Phe Leu Asp Pro Phe Asp Arg Cys Gly Leu Ile Val Ala Lys Tyr 145 150 155 160 Phe Ser Leu Pro Ser Val Val Phe Ala Arg Gly Ile Phe Cys His Tyr 165 170 175 Leu Glu Glu Gly Ala Gln Cys Pro Ala Pro Leu Ser Tyr Val Pro Arg 180 185 190 Leu Leu Leu Gly Phe Ser Asp Ala Met Thr Phe Lys Glu Arg Val Trp 195 200 205 Asn His Ile Met His Leu Glu Glu His Leu Phe Cys Pro Tyr Phe Phe 210 215 220 Lys Asn Val Leu Glu Ile Ala Ser Glu Ile Leu Gln Thr Pro Val Thr 225 230 235 240 Ala Tyr Asp Leu Tyr Ser His Thr Ser Ile Trp Leu Leu Arg Thr Asp 245 250 255 Phe Val Leu Glu Tyr Pro Lys Pro Val Met Pro Asn Met Ile Phe Ile 260 265 270 Gly Gly Ile Asn Cys His Gln Gly Lys Pro Val Pro Met Val Ser Tyr 275 280 285 Leu Pro Phe Ser Thr Leu Arg Ile Ile Trp Leu Trp Lys Leu Lys Asp 290 295 300 Phe Leu Gln Asn His Asn Leu Ser Phe Thr Phe Val Pro 305 310 315 13 930 DNA Homo sapiens CDS (1)...(930) 13 atg gct cgc aca ggg tgg acc agc ccc att ccc cta tgt gtt tct ctg 48 Met Ala Arg Thr Gly Trp Thr Ser Pro Ile Pro Leu Cys Val Ser Leu 1 5 10 15 ctg ctg acc tgt ggc ttt gct gag gca ggg aag ctg ctg gta gtg ccc 96 Leu Leu Thr Cys Gly Phe Ala Glu Ala Gly Lys Leu Leu Val Val Pro 20 25 30 atg gat ggg agt cac tgg ttc acc atg cag tcg gtg gtg gag aaa ctt 144 Met Asp Gly Ser His Trp Phe Thr Met Gln Ser Val Val Glu Lys Leu 35 40 45 atc ctc agg ggg cat gag gtg gtt gta gtc atg cca gag gtg agt tgg 192 Ile Leu Arg Gly His Glu Val Val Val Val Met Pro Glu Val Ser Trp 50 55 60 caa ctg gga aaa tca ctg aat tgc aca gtg aag act tac tca acc tca 240 Gln Leu Gly Lys Ser Leu Asn Cys Thr Val Lys Thr Tyr Ser Thr Ser 65 70 75 80 tac act ctg gag gat ctg gac cgg gaa ttc atg gat ttc gcc gat gct 288 Tyr Thr Leu Glu Asp Leu Asp Arg Glu Phe Met Asp Phe Ala Asp Ala 85 90 95 caa tgg aaa gca caa gta cga agt ttg ttt tct cta ttt ctg agt tca 336 Gln Trp Lys Ala Gln Val Arg Ser Leu Phe Ser Leu Phe Leu Ser Ser 100 105 110 tcc aat ggt ttt ttt aac tta ttt ttt tcg cat tgc agg agt ttg ttt 384 Ser Asn Gly Phe Phe Asn Leu Phe Phe Ser His Cys Arg Ser Leu Phe 115 120 125 aat gac cga aaa tta gta gaa tac tta aag gag agt tct ttt gat gcg 432 Asn Asp Arg Lys Leu Val Glu Tyr Leu Lys Glu Ser Ser Phe Asp Ala 130 135 140 gtg ttt ctt gat cct ttt gat gcc tgt gcg tta att gtt gcc aaa tat 480 Val Phe Leu Asp Pro Phe Asp Ala Cys Ala Leu Ile Val Ala Lys Tyr 145 150 155 160 ttc tcc ctc ccc tct gtg gtc ttc gcc agg gga ata ggt tgc cac tat 528 Phe Ser Leu Pro Ser Val Val Phe Ala Arg Gly Ile Gly Cys His Tyr 165 170 175 ctt gaa gaa ggt gca cag tgc cct gct cct ctt tcc tat gtc ccc aga 576 Leu Glu Glu Gly Ala Gln Cys Pro Ala Pro Leu Ser Tyr Val Pro Arg 180 185 190 att ctc tta ggg ttc tca gat gcc atg act ttc aag gag aga gta cgg 624 Ile Leu Leu Gly Phe Ser Asp Ala Met Thr Phe Lys Glu Arg Val Arg 195 200 205 aac cac atc atg cac ttg gag gaa cat tta ttt tgc cag tat ttt tcc 672 Asn His Ile Met His Leu Glu Glu His Leu Phe Cys Gln Tyr Phe Ser 210 215 220 aaa aat gcc cta gaa ata gcc tct gaa att ctc caa aca cct gtc aca 720 Lys Asn Ala Leu Glu Ile Ala Ser Glu Ile Leu Gln Thr Pro Val Thr 225 230 235 240 gca tat gat ctc tac agc cac aca tca att tgg ttg ttg cga aca gac 768 Ala Tyr Asp Leu Tyr Ser His Thr Ser Ile Trp Leu Leu Arg Thr Asp 245 250 255 ttt gtt ttg gac tat ccc aaa ccc gtg atg ccc aat atg atc ttc att 816 Phe Val Leu Asp Tyr Pro Lys Pro Val Met Pro Asn Met Ile Phe Ile 260 265 270 ggt ggt atc aac tgc cat cag gga aag cca ttg cct atg gta agt cac 864 Gly Gly Ile Asn Cys His Gln Gly Lys Pro Leu Pro Met Val Ser His 275 280 285 ctc tcc ttt agc aca tta gga ata atc ttg gct ttg gaa att aaa aaa 912 Leu Ser Phe Ser Thr Leu Gly Ile Ile Leu Ala Leu Glu Ile Lys Lys 290 295 300 aga ttc ctt act gaa ttg 930 Arg Phe Leu Thr Glu Leu 305 310 14 310 PRT Homo sapiens 14 Met Ala Arg Thr Gly Trp Thr Ser Pro Ile Pro Leu Cys Val Ser Leu 1 5 10 15 Leu Leu Thr Cys Gly Phe Ala Glu Ala Gly Lys Leu Leu Val Val Pro 20 25 30 Met Asp Gly Ser His Trp Phe Thr Met Gln Ser Val Val Glu Lys Leu 35 40 45 Ile Leu Arg Gly His Glu Val Val Val Val Met Pro Glu Val Ser Trp 50 55 60 Gln Leu Gly Lys Ser Leu Asn Cys Thr Val Lys Thr Tyr Ser Thr Ser 65 70 75 80 Tyr Thr Leu Glu Asp Leu Asp Arg Glu Phe Met Asp Phe Ala Asp Ala 85 90 95 Gln Trp Lys Ala Gln Val Arg Ser Leu Phe Ser Leu Phe Leu Ser Ser 100 105 110 Ser Asn Gly Phe Phe Asn Leu Phe Phe Ser His Cys Arg Ser Leu Phe 115 120 125 Asn Asp Arg Lys Leu Val Glu Tyr Leu Lys Glu Ser Ser Phe Asp Ala 130 135 140 Val Phe Leu Asp Pro Phe Asp Ala Cys Ala Leu Ile Val Ala Lys Tyr 145 150 155 160 Phe Ser Leu Pro Ser Val Val Phe Ala Arg Gly Ile Gly Cys His Tyr 165 170 175 Leu Glu Glu Gly Ala Gln Cys Pro Ala Pro Leu Ser Tyr Val Pro Arg 180 185 190 Ile Leu Leu Gly Phe Ser Asp Ala Met Thr Phe Lys Glu Arg Val Arg 195 200 205 Asn His Ile Met His Leu Glu Glu His Leu Phe Cys Gln Tyr Phe Ser 210 215 220 Lys Asn Ala Leu Glu Ile Ala Ser Glu Ile Leu Gln Thr Pro Val Thr 225 230 235 240 Ala Tyr Asp Leu Tyr Ser His Thr Ser Ile Trp Leu Leu Arg Thr Asp 245 250 255 Phe Val Leu Asp Tyr Pro Lys Pro Val Met Pro Asn Met Ile Phe Ile 260 265 270 Gly Gly Ile Asn Cys His Gln Gly Lys Pro Leu Pro Met Val Ser His 275 280 285 Leu Ser Phe Ser Thr Leu Gly Ile Ile Leu Ala Leu Glu Ile Lys Lys 290 295 300 Arg Phe Leu Thr Glu Leu 305 310 15 759 DNA Homo sapiens CDS (1)...(759) 15 atg gat ggg agt cac tgg ttc acc atg cag tcg gtg gtg gag aaa ctt 48 Met Asp Gly Ser His Trp Phe Thr Met Gln Ser Val Val Glu Lys Leu 1 5 10 15 atc ctc agg ggg cat gag gtg gtt gta gtc atg cca gag gtg agt tgg 96 Ile Leu Arg Gly His Glu Val Val Val Val Met Pro Glu Val Ser Trp 20 25 30 caa ctg gaa aga tca ctg aat tgc aca gtg aag act tac tca acc tcg 144 Gln Leu Glu Arg Ser Leu Asn Cys Thr Val Lys Thr Tyr Ser Thr Ser 35 40 45 tac act ctg gaa gat cag aac cgg gaa ttc atg gtt ttc gcc cat gct 192 Tyr Thr Leu Glu Asp Gln Asn Arg Glu Phe Met Val Phe Ala His Ala 50 55 60 caa tgg aaa gca cag gca caa agt ata ttt tct cta tta atg agt tca 240 Gln Trp Lys Ala Gln Ala Gln Ser Ile Phe Ser Leu Leu Met Ser Ser 65 70 75 80 tcc agt ggt ttt ctt gac tta ttt ttt tcg cat tgc agg agt ttg ttt 288 Ser Ser Gly Phe Leu Asp Leu Phe Phe Ser His Cys Arg Ser Leu Phe 85 90 95 aat gac cga aaa tta gta gaa tac tta aag gag agt tct ttt gat gca 336 Asn Asp Arg Lys Leu Val Glu Tyr Leu Lys Glu Ser Ser Phe Asp Ala 100 105 110 gtg ttt ctg gat cct ttt gat acc tgt ggc tta att gtt gct aaa tat 384 Val Phe Leu Asp Pro Phe Asp Thr Cys Gly Leu Ile Val Ala Lys Tyr 115 120 125 ttc tcc ctc ccc tct gtg gtc ttc acc agg gga ata ttt tgc cac cat 432 Phe Ser Leu Pro Ser Val Val Phe Thr Arg Gly Ile Phe Cys His His 130 135 140 ctt gaa gaa ggt gca cag tgc cct gct cct ctt tcc tat gtc ccc aat 480 Leu Glu Glu Gly Ala Gln Cys Pro Ala Pro Leu Ser Tyr Val Pro Asn 145 150 155 160 gat ctc tta ggg ttc tca gat gcc atg act ttc aag gag aga gta tgg 528 Asp Leu Leu Gly Phe Ser Asp Ala Met Thr Phe Lys Glu Arg Val Trp 165 170 175 aac cac atc gtg cac ttg gag gac cat tta ttt tgc cag tat ctt ttt 576 Asn His Ile Val His Leu Glu Asp His Leu Phe Cys Gln Tyr Leu Phe 180 185 190 aga aat gcc cta gaa ata gcc tct gaa att ctc caa acc cct gtc acg 624 Arg Asn Ala Leu Glu Ile Ala Ser Glu Ile Leu Gln Thr Pro Val Thr 195 200 205 gca tat gat ctc tac agt cac aca tca att tgg ttg ttg cga acg gac 672 Ala Tyr Asp Leu Tyr Ser His Thr Ser Ile Trp Leu Leu Arg Thr Asp 210 215 220 ttt gtt ttg gac tat ccc aaa ccc gtg atg ccc aac atg atc ttc att 720 Phe Val Leu Asp Tyr Pro Lys Pro Val Met Pro Asn Met Ile Phe Ile 225 230 235 240 ggt ggt atc aac tgt cat cag gga aag cca ttg cct atg 759 Gly Gly Ile Asn Cys His Gln Gly Lys Pro Leu Pro Met 245 250 16 253 PRT Homo sapiens 16 Met Asp Gly Ser His Trp Phe Thr Met Gln Ser Val Val Glu Lys Leu 1 5 10 15 Ile Leu Arg Gly His Glu Val Val Val Val Met Pro Glu Val Ser Trp 20 25 30 Gln Leu Glu Arg Ser Leu Asn Cys Thr Val Lys Thr Tyr Ser Thr Ser 35 40 45 Tyr Thr Leu Glu Asp Gln Asn Arg Glu Phe Met Val Phe Ala His Ala 50 55 60 Gln Trp Lys Ala Gln Ala Gln Ser Ile Phe Ser Leu Leu Met Ser Ser 65 70 75 80 Ser Ser Gly Phe Leu Asp Leu Phe Phe Ser His Cys Arg Ser Leu Phe 85 90 95 Asn Asp Arg Lys Leu Val Glu Tyr Leu Lys Glu Ser Ser Phe Asp Ala 100 105 110 Val Phe Leu Asp Pro Phe Asp Thr Cys Gly Leu Ile Val Ala Lys Tyr 115 120 125 Phe Ser Leu Pro Ser Val Val Phe Thr Arg Gly Ile Phe Cys His His 130 135 140 Leu Glu Glu Gly Ala Gln Cys Pro Ala Pro Leu Ser Tyr Val Pro Asn 145 150 155 160 Asp Leu Leu Gly Phe Ser Asp Ala Met Thr Phe Lys Glu Arg Val Trp 165 170 175 Asn His Ile Val His Leu Glu Asp His Leu Phe Cys Gln Tyr Leu Phe 180 185 190 Arg Asn Ala Leu Glu Ile Ala Ser Glu Ile Leu Gln Thr Pro Val Thr 195 200 205 Ala Tyr Asp Leu Tyr Ser His Thr Ser Ile Trp Leu Leu Arg Thr Asp 210 215 220 Phe Val Leu Asp Tyr Pro Lys Pro Val Met Pro Asn Met Ile Phe Ile 225 230 235 240 Gly Gly Ile Asn Cys His Gln Gly Lys Pro Leu Pro Met 245 250 17 735 DNA Homo sapiens CDS (1)...(735) 17 gaa ttt gaa gcc tac att aat gct tct gga gaa cat gga att gtg gtt 48 Glu Phe Glu Ala Tyr Ile Asn Ala Ser Gly Glu His Gly Ile Val Val 1 5 10 15 ttc tct ttg gga tca atg gtc tca gaa att cca gag aag aaa gct atg 96 Phe Ser Leu Gly Ser Met Val Ser Glu Ile Pro Glu Lys Lys Ala Met 20 25 30 gca att gct gat gct ttg ggc aaa atc cct cag aca gtc ctg tgg cgg 144 Ala Ile Ala Asp Ala Leu Gly Lys Ile Pro Gln Thr Val Leu Trp Arg 35 40 45 tac act gga acc cga cca tcg aat ctt gcg aac aac acg ata ctt gtt 192 Tyr Thr Gly Thr Arg Pro Ser Asn Leu Ala Asn Asn Thr Ile Leu Val 50 55 60 aag tgg cta ccc caa aac gat ctg ctt ggt cac ccg atg acc cgt gcc 240 Lys Trp Leu Pro Gln Asn Asp Leu Leu Gly His Pro Met Thr Arg Ala 65 70 75 80 ttt atc acc cat gct ggt tcc cat ggt gtt tat gaa agc ata tgc aat 288 Phe Ile Thr His Ala Gly Ser His Gly Val Tyr Glu Ser Ile Cys Asn 85 90 95 ggc gtt ccc atg gtg atg atg ccc ttg ttt ggt gat cag atg gac aat 336 Gly Val Pro Met Val Met Met Pro Leu Phe Gly Asp Gln Met Asp Asn 100 105 110 gca aag cgc atg gag act aag gga gct gga gtg acc ctg aat gtt ctg 384 Ala Lys Arg Met Glu Thr Lys Gly Ala Gly Val Thr Leu Asn Val Leu 115 120 125 gaa atg act tct gaa gat tta gaa aat gct cta aaa gca gtc atc aat 432 Glu Met Thr Ser Glu Asp Leu Glu Asn Ala Leu Lys Ala Val Ile Asn 130 135 140 gac aaa agt tac aag gag aac atc atg cgc ctc tcc agc ctt cac aag 480 Asp Lys Ser Tyr Lys Glu Asn Ile Met Arg Leu Ser Ser Leu His Lys 145 150 155 160 gac cgc ccg gtg gag ccg ctg gac ctg gcc gtg ttc tgg gtg gag ttt 528 Asp Arg Pro Val Glu Pro Leu Asp Leu Ala Val Phe Trp Val Glu Phe 165 170 175 gtg atg agg cac aag ggc gcg cca cac ctg cgc ccc gca gcc cac gac 576 Val Met Arg His Lys Gly Ala Pro His Leu Arg Pro Ala Ala His Asp 180 185 190 ctc acc tgg tac cag tac cat tcc ttg gac gtg att ggt ttc ctc ttg 624 Leu Thr Trp Tyr Gln Tyr His Ser Leu Asp Val Ile Gly Phe Leu Leu 195 200 205 gcc gtc gtg ctg aca gtg gcc ttc atc acc ttt aaa tgt tgt gct tat 672 Ala Val Val Leu Thr Val Ala Phe Ile Thr Phe Lys Cys Cys Ala Tyr 210 215 220 ggc tac cgg aaa tgc ttg ggg aaa aaa ggg cga gtt aag aaa gcc cac 720 Gly Tyr Arg Lys Cys Leu Gly Lys Lys Gly Arg Val Lys Lys Ala His 225 230 235 240 aaa tcc aag acc cat 735 Lys Ser Lys Thr His 245 18 245 PRT Homo sapiens 18 Glu Phe Glu Ala Tyr Ile Asn Ala Ser Gly Glu His Gly Ile Val Val 1 5 10 15 Phe Ser Leu Gly Ser Met Val Ser Glu Ile Pro Glu Lys Lys Ala Met 20 25 30 Ala Ile Ala Asp Ala Leu Gly Lys Ile Pro Gln Thr Val Leu Trp Arg 35 40 45 Tyr Thr Gly Thr Arg Pro Ser Asn Leu Ala Asn Asn Thr Ile Leu Val 50 55 60 Lys Trp Leu Pro Gln Asn Asp Leu Leu Gly His Pro Met Thr Arg Ala 65 70 75 80 Phe Ile Thr His Ala Gly Ser His Gly Val Tyr Glu Ser Ile Cys Asn 85 90 95 Gly Val Pro Met Val Met Met Pro Leu Phe Gly Asp Gln Met Asp Asn 100 105 110 Ala Lys Arg Met Glu Thr Lys Gly Ala Gly Val Thr Leu Asn Val Leu 115 120 125 Glu Met Thr Ser Glu Asp Leu Glu Asn Ala Leu Lys Ala Val Ile Asn 130 135 140 Asp Lys Ser Tyr Lys Glu Asn Ile Met Arg Leu Ser Ser Leu His Lys 145 150 155 160 Asp Arg Pro Val Glu Pro Leu Asp Leu Ala Val Phe Trp Val Glu Phe 165 170 175 Val Met Arg His Lys Gly Ala Pro His Leu Arg Pro Ala Ala His Asp 180 185 190 Leu Thr Trp Tyr Gln Tyr His Ser Leu Asp Val Ile Gly Phe Leu Leu 195 200 205 Ala Val Val Leu Thr Val Ala Phe Ile Thr Phe Lys Cys Cys Ala Tyr 210 215 220 Gly Tyr Arg Lys Cys Leu Gly Lys Lys Gly Arg Val Lys Lys Ala His 225 230 235 240 Lys Ser Lys Thr His 245 19 19 DNA Artificial Sequence Primer 19 tggtgtatcg attggtttt 19 20 23 DNA Artificial Sequence Primer 20 catatatctg gggctagtta atc 23 21 26 DNA Artificial Sequence Primer 21 acaaggtaat taagatgaag aaagca 26 22 20 DNA Artificial Sequence Primer 22 acctgagata gtggcttcct 20 23 20 DNA Artificial Sequence Primer 23 tttgtcttcc aattacatgc 20 24 24 DNA Artificial Sequence Primer 24 agtagatatg gaagcacttg taag 24 25 24 DNA Artificial Sequence Primer 25 tctcagtgac aaggtaatta agac 24 26 19 DNA Artificial Sequence Primer 26 cattgattgg ataaaggca 19 27 22 DNA Artificial Sequence Primer 27 aatttgggtt cttacatatc aa 22 28 18 DNA Artificial Sequence Primer 28 gagtgaggga ggacagag 18 29 21 DNA Artificial Sequence Primer 29 ataagtacac gccttctttt g 21 30 22 DNA Artificial Sequence Primer 30 gctgctttat acaatttgct ac 22 31 22 DNA Artificial Sequence Primer 31 cgcctacgta tcatagcagt ta 22 32 22 DNA Artificial Sequence Primer 32 ggaaagaaat ttgaaatgca ac 22 33 20 DNA Artificial Sequence Primer 33 tctttccgcc tactgtatca 20 34 20 DNA Artificial Sequence Primer 34 ttcaagaagg gcagttttat 20 35 17 DNA Artificial Sequence Primer 35 ctctggcagg agcaaag 17 36 20 DNA Artificial Sequence Primer 36 atacacacct gggatagtgg 20 37 22 DNA Artificial Sequence Primer 37 ggtaattaag atgaagaaag ca 22 38 19 DNA Artificial Sequence Primer 38 ctgagatagt ggcttcctg 19 39 17 DNA Artificial Sequence Primer 39 gtggctcaat gacaagg 17 40 23 DNA Artificial Sequence Primer 40 atatggaagc acttgtaagt aaa 23 41 22 DNA Artificial Sequence Primer 41 ttaagacgaa ggaaacaatt ct 22 42 19 DNA Artificial Sequence Primer 42 acctgagata gtggcttcc 19 43 20 DNA Artificial Sequence Primer 43 atcaaagggt aaaattcaga 20 44 18 DNA Artificial Sequence Primer 44 ggcagtccaa aagaaata 18 45 18 DNA Artificial Sequence Primer 45 ttttgagggc aggttcta 18 46 22 DNA Artificial Sequence Primer 46 aatgggacaa atgtaaatga ta 22 47 17 DNA Artificial Sequence Primer 47 ttctctcatg gctcgca 17 48 24 DNA Artificial Sequence Primer 48 atgtcaaatc acaattcagt aagg 24 49 20 DNA Artificial Sequence Primer 49 ccgcctactg tatcatagca 20 50 21 DNA Artificial Sequence Primer 50 caacgaaatg tcaaatcaca g 21 51 17 DNA Artificial Sequence Primer 51 ctctggcagg agcaaag 17 52 17 DNA Artificial Sequence Primer 52 acagtgggca gagacag 17 53 18 DNA Artificial Sequence Primer 53 gtggtttatt ccccgtat 18 54 20 DNA Artificial Sequence Primer 54 atacacacct gggatagtgg 20 55 22 DNA Artificial Sequence Primer 55 ggtaattaag atgaagaaag ca 22 56 18 DNA Artificial Sequence Primer 56 gaaatggcat aggttgtc 18 57 17 DNA Artificial Sequence Primer 57 ggccacactc aactgta 17 58 20 DNA Artificial Sequence Primer 58 ctcaaaaaaa acacagtagg 20 59 18 DNA Artificial Sequence Primer 59 actttttctg ccccttat 18 60 23 DNA Artificial Sequence Primer 60 atatggaagc acttgtaagt aaa 23 61 22 DNA Artificial Sequence Primer 61 ttaagacgaa ggaaacaatt ct 22 62 17 DNA Artificial Sequence Primer 62 aatggcatac gttgtca 17 63 19 DNA Artificial Sequence Primer 63 agaatggcaa ttatgaaca 19 64 18 DNA Artificial Sequence Primer 64 tgtgtgccct taaagtct 18 65 19 DNA Artificial Sequence Primer 65 agaatggcaa ttatgaaca 19 66 19 DNA Artificial Sequence Primer 66 acctgagata gtggcttcc 19 67 17 DNA Artificial Sequence Primer 67 ctctggctct gtcctac 17 68 19 DNA Artificial Sequence Primer 68 acctgagata gtggcttcc 19 69 20 DNA Artificial Sequence Primer 69 atcaaagggt aaaattcaga 20 70 18 DNA Artificial Sequence Primer 70 cagcagcttg tcacctac 18 71 20 DNA Artificial Sequence Primer 71 aatttgcttt tgaaagaatc 20 72 18 DNA Artificial Sequence Primer 72 ggtaggccca aatactca 18 73 20 DNA Artificial Sequence Primer 73 aatttgcttt tgaaagaatc 20 74 18 DNA Artificial Sequence Primer 74 ggcagtccaa aagaaata 18 75 18 DNA Artificial Sequence Primer 75 ttttgagggc aggttcta 18 76 18 DNA Artificial Sequence Primer 76 cacctctggc atgactac 18 77 19 DNA Artificial Sequence Primer 77 ttgcaggagt ttgtttaat 19 78 22 DNA Artificial Sequence Primer 78 aatgggacaa atgtaaatga ta 22 79 19 DNA Artificial Sequence Primer 79 cattgcagga gtttgttta 19 80 20 DNA Artificial Sequence Primer 80 catctgagaa ccctaagaga 20 81 20 DNA Artificial Sequence Primer 81 agaaatagcc tctgaaattc 20 82 24 DNA Artificial Sequence Primer 82 atgtcaaatc acaattcagt aagg 24 83 20 DNA Artificial Sequence Primer 83 ccgcctactg tatcatagca 20 84 21 DNA Artificial Sequence Primer 84 gagtgtacga ggttgagtaa g 21 85 21 DNA Artificial Sequence Primer 85 attttgccag tatcttttta g 21 86 21 DNA Artificial Sequence Primer 86 caacgaaatg tcaaatcaca g 21 87 27 DNA Homo sapiens 87 catcagagac agagcatttt acacctt 27 88 26 DNA Homo sapiens 88 ggacctattg agccctgcat ctgtct 26 89 25 DNA Homo sapiens 89 ggttcccctg ccgcggctgg ccaca 25 90 22 DNA Homo sapiens 90 gccctgggct gaaagtggaa ag 22 91 23 DNA Homo sapiens 91 atgcgggagg ccttgcggga gct 23 92 24 DNA Homo sapiens 92 ctctgcgcgg cggtgctggc taag 24 93 25 DNA Homo sapiens 93 taccccaggc caatcatgcc caaca 25 94 27 DNA Homo sapiens 94 tccaggcaaa atacttttta aaaaatg 27 95 23 DNA Homo sapiens 95 agcatgcggg aggcctcgcg gga 23 96 21 DNA Homo sapiens 96 gcgggagctc catgcgagag g 21 97 25 DNA Homo sapiens 97 tggtggtcct caccccggag gtgaa 25 98 26 DNA Homo sapiens 98 tacatcaaag aggagaactt tttcac 26 99 26 DNA Homo sapiens 99 tgatcaggca cctgaatgct acttcc 26 100 21 DNA Homo sapiens 100 acctctgcgg ggcggtgctg g 21 101 23 DNA Homo sapiens 101 aagaacatgc tttaccctct ggc 23 102 18 DNA Homo sapiens 102 ctctggctct gtcctacc 18 103 24 DNA Homo sapiens 103 tcctaccttt gctatgctgt ttct 24 104 17 DNA Homo sapiens 104 tgtcagtggt ggatatt 17 105 16 DNA Homo sapiens 105 ggtggatctt ctcagc 16 106 13 DNA Homo sapiens 106 tcagctatgc atc 13 107 21 DNA Homo sapiens 107 gcatccgtgt ggctgttccg a 21 108 20 DNA Homo sapiens 108 tggctgttcc gacgggactt 20 109 16 DNA Homo sapiens 109 gggacttcgt gatgga 16 110 23 DNA Homo sapiens 110 gtgatggact accccaggcc gat 23 111 25 DNA Homo sapiens 111 cctgcctcct tcgcgcattt cagag 25 112 25 DNA Homo sapiens 112 gcgatcattc ctgactgctc ctcag 25 113 22 DNA Homo sapiens 113 ccctggagca tgcattcagc ag 22 114 23 DNA Homo sapiens 114 cattcagcag cagcccagac cct 23 115 23 DNA Homo sapiens 115 tacttcttcc acgtactata tta 23 116 24 DNA Homo sapiens 116 ggcctccttc cactatatgt gtgt 24 117 21 DNA Homo sapiens 117 ggagagagta cggaaccaca t 21 118 23 DNA Homo sapiens 118 tcaatttggt tattgcgaac tga 23 119 22 DNA Homo sapiens 119 caggggaata gcttgccact at 22 120 24 DNA Homo sapiens 120 tgttgcgaac ggactttgtt ttgg 24 121 22 DNA Homo sapiens 121 ttcaccatgc aatcggtggt gg 22 122 25 DNA Homo sapiens 122 ctagaaatag cttctgaaat tctcc 25 123 24 DNA Homo sapiens 123 cggcatatga tatctacagt caca 24 124 24 DNA Homo sapiens 124 tcaatttggt tgctgcgaac ggac 24 

What is claimed is:
 1. An isolated nucleic acid molecule comprising a UGT1 sequence polymorphism of SEQ ID NOS: 87-124, as part of other than a naturally occurring chromosome.
 2. A nucleic acid probe for detection of UGT1 locus polymorphisms, comprising a polymorphic sequence of SEQ ID NOS:87-124.
 3. A nucleic acid probe according to claim 2, wherein said probe is conjugated to a detectable marker.
 4. An array of oligonucleotides comprising: two or more probes for detection of UGT1 locus polymorphisms, said probes comprising at least one form of a polymorphic sequences of SEQ ID NOS:87-124.
 5. A method for detecting in an individual a polymorphism in UGT1 metabolism of a substrate, the method comprising: analyzing the genome of said individual for the presence of at least one UGT1 polymorphism of SEQ ID NOS:87-124; wherein the presence of said predisposing polymorphism is indicative of an alteration in UGT1 expression or activity.
 6. A method according to claim 5, wherein said analyzing step comprises detection of specific binding between the genomic DNA of said individual with an array of oligonucleotides comprising: two or more probes for detection of UGT1 locus polymorphisms, said probes comprising at least one form of a polymorphic sequence of SEQ ID NOS:87-124.
 7. A method according to claim 5, wherein said alteration in UGT1 expression is tissue specific.
 8. A method according to claim 5, wherein said alteration in UGT1 expression is in response to a UGT1 modifier.
 9. A method according to claim 8, wherein said modifier induces UGT1 expression.
 10. A method according to claim 8, wherein said modifier inhibits UGT1 expression. 